CL ¬eo|thcore

lure s|mp||c|tg íor
togged prote|ns
The journeg írom o torget gene to pur|ned prote|n 3
C$T-togged prote|ns
lntroduct|on to the C$T íus|on sgstem 4
pCL× vectors 5
^pp||cot|ons 6
Tog c|eovoge enzgmes l3
^pp||cot|ons l4
0etect|on oí C$T-togged prote|ns l7
0rder|ng |níormot|on l9
¬|st|d|ne-togged prote|ns
lntroduct|on to h|st|d|ne-togged prote|ns 20
^pp||cot|ons 2l
0rder|ng |níormot|on 3l
Strep-tog ll prote|ns
lntroduct|on to Strep-tog ll prote|ns 32
^pp||cot|ons 33
0rder|ng |níormot|on 36
l3l-togged prote|ns
lntroduct|on to l3l-togged prote|ns 38
^pp||cot|ons 39
0rder|ng |níormot|on 43
0uo|-togged prote|ns
lntroduct|on to duo|-togged prote|ns 44
^pp||cot|ons 45
Contents
The journeg írom o torget
gene to pur|ned prote|n
The use oí recomb|nont prote|ns hos |ncreosed greot|g |n recent geors,
os hos the weo|th oí techn|ques ond products used íor the|r omp||ncot|on
ond pur|ncot|on. The odvontoges oí us|ng o recomb|nont togged prote|n
to íoc|||tote pur|ncot|on ond detect|on ore now w|de|g recogn|zed. ln
genero|, the nrst step |n o prote|n express|on workfow |nvo|ves c|on|ng oí
the torget gene |nto on oppropr|ote express|on vector. Th|s |s ío||owed bg
the tronsíormot|on oí the express|on vector |nto o su|tob|e host sgstem
íor subsequent express|on ono|gs|s.
long host sgstems ore ovo||ob|e |nc|ud|ng bocter|o, geost, p|onts,
n|omentous íung|, |nsect or mommo||on ce||s grown |n cu|ture, ond
tronsgen|c on|mo|s or p|onts. Loch host sgstem hos |ts own odvontoges
ond d|sodvontoges, ond |t |s |mportont to cons|der these beíore the nno|
se|ect|on oí o host. ^íter |n|t|o| screen|ng ond |dent|ncot|on oí opt|mum
express|on cond|t|ons íor gour port|cu|or prote|n, gou con sco|e-up the
pur|ncot|on process to obto|n |orge omounts oí the torget prote|n íor
downstreom opp||cot|ons such os íunct|ono| ond structuro| stud|es.
The pur|ncot|on oí togged prote|ns |s re|ot|ve|g s|mp|e ond soves t|me
due to the h|gh spec|nc|tg between the tog on the expressed prote|n ond
the ||gond on the oínn|tg med|um. \ou get h|gh pur|tg |n o s|ng|e step-
oínn|tg pur|ncot|on tgp|co||g g|ves up to 95% pur|tg. further pur|ncot|on
steps mog be necessorg |í gou requ|re greoter pur|tg.
Cloning Cell culture Screening
Scale-up
purification
Structural
& functional
studies
Lead design
3
4
C$T-togged prote|ns
C|utoth|one $-tronsíerose |C$Tl Cene fus|on $gstem |s o versot||e
sgstem íor the express|on, pur|ncot|on, ond detect|on oí C$T-togged
prote|ns produced |n E. coli. Tgp|co| íeotures oí C$T-togged prote|ns
|nc|ude h|gh b|nd|ng spec|nc|tg to g|utoth|one ||gonds on C|utoth|one
$ephorose¯ chromotogrophg med|o, resu|t|ng |n greoter thon 90%
pur|tg |n one step oí the e|uted torget mo|ecu|e. The C$T tog mog
o|so |ncreose the so|ub|||tg ond stob|||tg oí the torget prote|n. C$T |s
re|ot|ve|g |orge w|th o re|ot|ve mo|ecu|or moss |l
r
l oí 26 000.
^ spec|nc c|eovoge sequence o||ows s|mp|e removo| oí the C$T tog
w|th the use oí d|ííerent proteoses oíter pur|ncot|on.
lur|ncot|on oí C$T-togged prote|ns con be períormed under verg
m||d cond|t|ons, wh|ch preserves the íunct|on ond ont|gen|c|tg oí the
torget prote|n. C$T togs ore oíten used to comp|ement h|st|d|ne togs
or os on o|ternot|ve when h|st|d|ne togs do not g|ve o so|ub|e prote|n
dur|ng express|on.
G
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pCL× vectors produce h|gh
g|e|ds oí togged prote|ns
C$T Cene fus|on $gstem |ncorporotes pCL× p|osm|ds íor
|nduc|b|e, h|gh-|eve| |ntroce||u|or express|on oí genes or
gene-írogments os íus|ons w|th Schistosoma japonicum
C$T. Lxpress|on |n E. coli g|e|ds togged prote|ns |n the
cgtop|osm w|th the C$T mo|etg ot the om|no term|nus ond
the prote|n oí |nterest ot the corboxg| term|nus.
Th|rteen pCL× vectors ore ovo||ob|e. N|ne oí the vectors
hove on exponded mu|t|p|e c|on|ng s|te |lC$l thot conto|ns
s|x restr|ct|on s|tes. The exponded lC$ íoc|||totes the
un|d|rect|ono| c|on|ng oí c0N^ |nserts obto|ned írom
||bror|es constructed us|ng mong ovo||ob|e |ombdo vectors.
CL ¬eo|thcore oííers pCL× vectors w|th encoded recogn|t|on
sequences íor s|te-spec|nc c|eovoge bg lre$c|ss|on¯
lroteose, Thromb|n proteose, ond foctor ×o proteose |see
pCL× vector sequence mop on th|s pogel.
pGEX-1T (27-4805-01)
pGEX-6P-1(27-4597-01)
EcoR I
Sma I
Sal I
Xho I
Not I BamH I
PreScission Protease
Leu Glu Val Leu Phe Gln Gly Pro Leu Gly Ser Pro Glu Phe Pro Gly Arg Leu Glu Arg Pro His
CTG GAA GTT CTG TTC CAG GGG CCC CTG GGA TCC CCG GAA TTC CCG GGT CGA CTC GAG CGG CCG CAT

pGEX-6P-2 (27-4598-01)
BamH I
PreScission Protease
Leu Glu Val Leu Phe Gln Gly Pro Leu Gly Ser Pro Gly Ile Pro Gly Ser Thr Arg Ala Ala Ala Ser
CTG GAA GTT CTG TTC CAG GGG CCC CTG GGA TCC CCAGGA ATT CCC GGG TCG ACT CGA GCG GCC GCA TCG

EcoR I
Sma I
Sal I
Xho I
Not I
pGEX-6P-3 (27-4599-01)
BamH I
PreScission Protease
Leu Glu Val Leu Phe Gln Gly Pro Leu Gly Ser Pro Asn Ser Arg Val Asp Ser Ser Gly Arg
CTG GAA GTT CTG TTC CAG GGG CCC CTG GGA TCC CCG AAT TCC CGG GTC GAC TCG AGC GGC CGC

EcoR I
Sma I
Sal I
Xho I
Not I
BamH I EcoR I
Sma I
Sal I
Xho I
Not I
BamH I EcoR I
Sma I
Sal I
Xho I
Not I
BamH I EcoR I
Sma I
Sal I
Xho I
Not I
BamH I EcoR I
Sma I
Sal I
Xho I
Not I
BamH I EcoR I
Sma I
Sal I
Xho I
Not I
BamH I EcoR I
Sma I
Sal I
Xho I
Not I
EcoR I
CTG GTT CCG CGT GGA TCC CCG GAA TTC ATC GTG ACT GAC TGA CGA
BamH I
Leu Val Pro Arg Gly Ser Pro Glu Phe Ile Val Thr Asp
Thrombin Protease
Stop codons

pGEX
~4900 bp
pBR322
ori
Bal I
BspM I
Ptac
c

a

l
I
q

Nar I
EcoR V
BssH II
BstE II
Mlu I
Apa I
Tth111 I
Aat II
Pst I
p4.5
AlwN I
pSj10 Bam7Stop7
pGEX-4T-2 (27-4581-01)
pGEX-5X-1 (27-4584-01)
pGEX-5X-2 (27-4585-01)
pGEX-5X-3 (27-4586-01)
pGEX-4T-1 (27-4580-01)
pGEX-4T-3 (27-4583-01)
pGEX-3X (27-4803-01)
pGEX-2TK (27-4587-01)
Leu Val Pro Arg Gly Ser Pro Gly Ile Pro Gly Ser Thr Arg Ala Ala Ala Ser
CTG GTT CCG CGT GGA TCC CCA GGA ATT CCC GGG TCG ACT CGA GCG GCC GCA TCG TGA
Stop codon
Ile Glu Gly Arg Gly Ile Pro Glu Phe Pro Gly Arg Leu Glu Arg Pro His Arg Asp
ATC GAA GGT CGT GGG ATC CCC GAA TTC CCG GGT CGA CTC GAG CGG CCG CAT CGT GAC TGA
Stop codons
Ile Glu Gly Arg Gly Ile Pro Gly Ile Pro Gly Ser Thr Arg Ala Ala Ala Ser
ATC GAA GGT CGT GGG ATC CCC GGA ATT CCC GGG TCG ACT CGA GCG GCC GCA TCG TGA
Stop codon
Ile Glu Gly Arg Gly Ile Pro Arg Asn Ser Arg Val Asp Ser Ser Gly Arg Ile Val Thr Asp
ATC GAA GGT CGT GGG ATC CCC AGG AAT TCC CGG GTC GAC TCG AGC GGC CGC ATC GTG ACT GAC TGA
Stop codons
Leu Val Pro Arg Gly Ser Pro Glu Phe Pro Gly Arg Leu Glu Arg Pro His Arg Asp
CTG GTT CCG CGT GGA TCC CCG GAA TTC CCG GGT CGA CTC GAG CGG CCG CAT CGT GAC TGA
Stop codons
Leu Val Pro Arg Gly Ser Pro Asn Ser Arg Val Asp Ser Ser Gly Arg Ile Val Thr Asp
CTG GTT CCG CGT GGA TCC CCG AAT TCC CGG GTC GAC TCG AGC GGC CGC ATC GTG ACT GAC TGA
Stop codons
ATC GAA GGT CGT GGG ATC CCC GGG AAT TCA TCG TGA CTG ACT GAC
Ile Glu Gly Arg Gly Ile Pro Gly Asn Ser Ser
Stop codons
Leu Val Pro Arg Gly Ser Arg Arg Ala Ser Val
Kinase
CTG GTT CCG CGT GGA TCT CGT CGT GCA TCT GTT GGA TCC CCG GGA ATT CAT CGT GAC TGA
Stop codons
Thrombin Protease

Thrombin Protease

Thrombin Protease

Thrombin Protease

Factor Xa Protease

Factor Xa Protease

Factor Xa Protease

Factor Xa Protease

EcoR I BamH I
Sma I
EcoR I BamH I
Sma I
pGEX-2T (27-4801-01)
CTG GTT CCG CGT GGA TCC CCG GGA ATT CAT CGT GAC TGA CTG ACG
Leu Val Pro Arg Gly Ser Pro Gly Ile His Arg Asp
Stop codons
EcoR I
Thrombin Protease

BamH I
Sma I
g
lu
ta
th
io
n
e S-transferase
A
m
p
r
Lven though stop codons |n o|| three íromes ore not dep|cted |n
th|s mop, o|| th|rteen vectors hove stop codons |n o|| three íromes
downstreom írom the mu|t|p|e c|on|ng s|te.
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lur|ncot|on oí
C$T-togged prote|ns
0|ííerent C|utoth|one $ephorose chromotogrophg med|o ore ovo||ob|e |n servero| íormots. The med|o vorg |n the|r períormonce
porometers, ond the d|ííerent íormots prov|de opt|ons íor sco|e ond conven|ence.
Conto|ns C|utoth|one $ephorose ¬|gh leríormonce
Conto|ns C|utoth|one $ephorose 4 fost f|ow
Conto|ns C|utoth|one $ephorose 43
No
v||| gou use on outomoted pur|ncot|on sgstem such os ^KT^des|gn¯` \es
C$Tlrep ff l6/l0
Crov|tg-fow co|umns
No
C|utoth|one $ephorose 43
|lrepocked d|sposob|e co|umnl
\es
3otch
No
3u|k C$T lur|ncot|on lodu|e
¯
lrepocked co|umns
C$T $p|nTrop lur|ncot|on lodu|e
¯
C$T lu|t|Trop 43
C|utoth|one $ephorose 43
C$T lu|t|Trop ff
Red|lock C$T lur|ncot|on lodu|e
¯
96-we|| p|ote
$p|n co|umn
C|utoth|one $ephorose 4 fost f|ow
C$Trop ff
$gr|nge
lrepocked co|umns
C$Trop ¬l
C|utoth|one $ephorose ¬|gh leríormonce
\es No
$co|ob|||tg/h|gh fow rotes
lrepocked co|umns
\es
^mount oí C$T-togged prote|n
< 50 mg > 50 mg
Glutathione Sepharose
C$Trop 43
¬|gh reso|ut|on
vhot |s most |mportont to gou`
3uííers |nc|uded`
No
\es
* Purification modules also contain buffers
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$creen|ng opt|mo|
cond|t|ons íor b|nd|ng
Products featured: GST MultiTrap FF and GST MultiTrap 4B, ExcelGel SDS Gradient 8–18,
Multiphor II Electrophoresis System, LMW-SDS Marker Kit
lrote|ns ore expressed íor íunct|ono| ond structuro| stud|es ond one port oí the eor|g screen|ng phose |s to nnd out opt|mo| cond|t|ons
íor b|nd|ng ond e|ut|on oí the torget prote|n.
The eííect oí d|ííerent |ncubot|on t|mes íor the b|nd|ng oí o C$T-togged prote|n somp|e wos |nvest|goted to och|eve the best g|e|d on
C$T lu|t|Trop¯ ff ond C$T lu|t|Trop 43 96-we|| n|ter p|otes. E. coli conto|n|ng C$T-h|ppoco|c|n wos chem|co||g |gsed ond son|coted
pr|or to opp||cot|on oí the unc|or|ned somp|e d|rect|g to the we||s.
96-well filter plates: C$T lu|t|Trop ff ond C$T lu|t|Trop 43
Sample: 300 µ| oí E. coli 3L2l |gsote conto|n|ng C$T-togged h|ppoco|c|n,
l
r
45 000
Sample preparation: Chem|co| |gs|s ond son|cot|on
Binding buffer: l0 ml sod|um phosphote, l40 ml NoC|, p¬ 8.0
Elution buffer: 50 ml Tr|s-¬C|, l0 ml reduced g|utoth|one, p¬ 8.0
Elution method: Centr|íugot|on
SDS-PAGE
500
450
400
350
300
250
200
0 20 40 60 80 100 120 140
Yield GST-hippocalcin, GST MultiTrap FF
Yield GST-hippocalcin, GST MultiTrap 4B
Incubation time (min)
A
v
e
r
a
g
e

y
i
e
l
d

o
f

e
l
u
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d

p
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e
i
n

(
μ
g
)
97
66
45
30
20
14
(M
r
× 10
3
)
GST-hippocalcin
L
M
W

m
a
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k
e
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s
S
t
a
r
t

m
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F
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o
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h
E
l
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a
t
e
F
l
o
w
t
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r
o
u
g
h
E
l
u
a
t
e
F
l
o
w
t
h
r
o
u
g
h
E
l
u
a
t
e
F
l
o
w
t
h
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o
u
g
h
E
l
u
a
t
e
F
l
o
w
t
h
r
o
u
g
h
E
l
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F
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o
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h
E
l
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t
e
120 min 60 min 30 min 10 min 3 min 0 min
Summary
The opt|mum t|me determ|ned íor the b|nd|ng oí C$T-h|ppoco|c|n wos 3 to l5 m|n w|th both C$T lu|t|Trop ff ond
C$T lu|t|Trop 43. The g|e|d wos ~350 µg us|ng both 96-we|| p|otes.
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$o|ub|||zot|on screen|ng
stroteg|es íor C$T-togged
membrone prote|ns
Products featured: GST SpinTrap Purification Module, GST Detection Module
$o|ub|||zot|on |s one oí the most cr|t|co| stoges oí the extroct|on oí membrone prote|ns. Lííect|ve so|ub|||zot|on ensures h|gh g|e|ds oí
b|o|og|co||g oct|ve membrone prote|n ond poves the wog íor successíu| pur|ncot|on. for eííect|ve so|ub|||zot|on oí o torget membrone
prote|n, |t |s oíten necessorg to screen severo| detergents beíore se|ect|ng the best detergent íor o port|cu|or torget prote|n ond |ts
pur|ncot|on strotegg.
C$T $p|nTrop¯ co|umns were used íor rop|d, s|mu|toneous screen|ng oí s|x d|ííerent detergents íor o C$T-membrone prote|n |C$T-ecoKchl.
96-well plate
l. The eííect oí d|ííerent detergents on the enzgmot|c
oct|v|tg oí o pur|ned C$T-prote|n wos meosured |n o
l-ch|oro-2,4-d|n|trobenzene |C0N3l ossog |n o 96-we||
p|ote íormot us|ng C$T 0etect|on lodu|e.
2. The membrone prote|ns were so|ub|||zed |n d|ííerent
detergents ond concentrot|ons thot do not oííect the
oct|v|tg oí the C$T tog.
3. The so|ub|||zed C$T-togged membrone prote|ns were
pur|ned us|ng C$T $p|nTrop lur|ncot|on lodu|e.

^no|gze g|e|d bg $0$-l^CL ond
oct|v|tg oí membrone prote|n.
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Column: C$T $p|nTrop
Sample: 500 µ| oí C$T-togged ecoKch so|ub|||zed l.l0 |w/vl |n
5% 00l, β0C, C¬^l$, Tween¯ 20, Tr|ton¯ ×-l00, or $orcosg|
Binding buffer: l3$, p¬ 7.5
Elution buffer: l3$, 0.2% detergent, l0 ml reduced g|utoth|one, p¬ 8.0
SDS-PAGE
DDM
OG
CHAPS Tween 20
Triton
X-100 Sarcosyl
GST-ecoKch
94
67
43
(M
r
× 10
3
)
L
M
W

m
a
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k
e
r
s
F
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o
u
g
h
E
l
u
a
t
e

f
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o
m

G
S
T

S
p
i
n
T
r
a
p
F
l
o
w
t
h
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o
u
g
h
E
l
u
a
t
e

f
r
o
m

G
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T

S
p
i
n
T
r
a
p
F
l
o
w
t
h
r
o
u
g
h
E
l
u
a
t
e

f
r
o
m

G
S
T

S
p
i
n
T
r
a
p
F
l
o
w
t
h
r
o
u
g
h
E
l
u
a
t
e

f
r
o
m

G
S
T

S
p
i
n
T
r
a
p
F
l
o
w
t
h
r
o
u
g
h
E
l
u
a
t
e

f
r
o
m

G
S
T

S
p
i
n
T
r
a
p

F
l
o
w
t
h
r
o
u
g
h
E
l
u
a
t
e

f
r
o
m

G
S
T

S
p
i
n
T
r
a
p
0.8
0.6
0.4
0.2
0
Solubilized material
Flowthrough
Eluate
A
c
t
i
v
i
t
y

(
A
3
4
0
/
m
i
n
*
m
l
)
D
D
M

O
G
C
H
A
P
S
T
w
e
e
n

2
0
T
r
it
o
n

X
-
1
0
0
S
a
r
c
o
s
y
l
Summary
The g|e|d oí C$T-ecoKch |n the so|ub|||zot|on |doto not shownl ond pur|í|cot|on steps wos h|ghest w|th 00l ond sorcosg|. Note
thot sorcosg| greot|g decreoses C$T oct|v|tg |n C0N3 ossogs thereíore the resu|ts do not reí|ect the true omounts oí C$T-togged
prote|n present. Lnzgme oct|v|tg oíter pur|í|cot|on wos h|ghest w|th 00l. Th|s shows o s|mp|e ond rop|d method íor se|ect|ng
opt|mum cond|t|ons íor membrone prote|n so|ub|||zot|on.
Acknowledgements: 0. 3|rse, 0eportment oí 3|ochem|strg ond 3|ophgs|cs, $tockho|m Un|vers|tg, $weden
G
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p
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l0
Línc|ent one-step pur|ncot|on
Products featured: GSTrap FF, ÄKTAexplorer, ExcelGel SDS Gradient 8–18, Multiphor II Electrophoresis System,
LMW-SDS Marker Kit
lur|ncot|on oí C$T-togged prote|ns con oíten be och|eved |n o s|ng|e step us|ng conven|ent prepocked co|umns comb|ned w|th
o sgr|nge, pump, or chromotogrophg sgstem. ln th|s exomp|e, o C$Trop¯ ff l m| co|umn wos used to pur|íg o C$T-togged
so|ub|e receptor subun|t to o h|gh degree oí pur|tg |n o s|ng|e step. ^ preprogrommed UNlC0RN¯ method temp|ote wos used
|n ^KT^exp|orer¯ to prov|de o stondord pur|ncot|on protoco| thot con e|ther be ío||owed exoct|g or mod|ned to su|t gour un|que
requ|rements. The e|uted íroct|on wos ono|gzed bg ge| e|ectrophores|s ío||owed bg s||ver sto|n|ng.
Column: C$Trop ff l m|
Sample: 8 m| oí cgtop|osm|c extroct írom E. coli express|ng o C$T-togged prote|n
Binding buffer: l3$, p¬ 7.3
Elution buffer: 50 ml Tr|s-¬C|, l0 ml reduced g|utoth|one, p¬ 8.0
Flow: l m|/m|n
System: ^KT^exp|orer
SDS-PAGE
0
20
40
60
80
100
%
Elution
buffer
0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
ml
Wash
Elution
buffer
2.7 mg
pure
GST-
tagged
protein
5.0 10.0 15.0 20.0
A
280
97
66
45
30
20
14
(M
r
× 10
3
)
L
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Summary
C$Trop ff ond o preprogrommed method temp|ote |n ^KT^exp|orer were used to produce h|gh|g pure prote|n |n o s|ng|e
pur|í|cot|on step.
G
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ll
Summary
The odd|t|on oí o second pur|í|cot|on step eííect|ve|g removed oggregotes írom the C$T-h|ppoco|c|n somp|e resu|t|ng |n the
recoverg oí h|gh|g pure torget prote|n.
Unottended two-step
outomoted pur|ncot|on
Products featured: GSTrap 4B, HiLoad 16/60 Superdex 200 pg, ÄKTAxpress, ExcelGel SDS Gradient 8–18,
Multiphor II Electrophoresis System, LMW-SDS Marker Kit
^íter the |n|t|o| pur|ncot|on oí o C$T-togged prote|n us|ng C|utoth|one $ephorose products, íree cgste|ne groups on the C$T tog con
reoct w|th eoch other to couse oggregotes desp|te the presence oí o reduc|ng ogent such os d|th|othre|to| |00Tl.
Runn|ng o second pur|ncot|on step us|ng ge| n|trot|on chromotogrophg prov|des on eínc|ent method íor seporot|ng prote|n oggregotes
írom the oct|ve torget prote|n. ^dd|t|ono| benents oí such o po||sh|ng step |nc|ude the removo| oí other contom|nonts ond buííer exchonge.
^KT^xpress¯ prov|des on outomoted so|ut|on íor unottended, mu|t|step pur|ncot|on oí oínn|tg-togged prote|ns. ^ggregotes oí C$T-
h|ppoco|c|n were successíu||g removed us|ng on outomoted two-step pur|ncot|on method temp|ote |n ^KT^xpress. The two-step method
compr|sed oínn|tg chromotogrophg |^Cl ond ge| n|trot|on |Cfl pur|ncot|on. The g|e|d oí e|uted C$T-h|ppoco|c|n wos meosured bg
obsorbonce ot 280 nm ond the pur|tg wos ono|gzed bg ge| e|ectrophores|s.
Columns: C$Trop 43 l m| |^Cl ond ¬|Lood¯ l6/60 $uperdex¯ 200 pg, l20 m| |Cfl
Sample: C|or|ned E. coli |gsote conto|n|ng expressed C$T-h|ppoco|c|n, l
r
43 000
Sample volume: 5 m|
Binding buffer (AC): l0 ml sod|um phosphote, l40 ml NoC|, 20 ml 0TT, p¬ 7.4
Elution buffer (AC): 50 ml Tr|s-¬C|, 20 ml g|utoth|one, 20 ml 0TT, p¬ 8.0
Buffer (GF): l0 ml sod|um phosphote, l40 ml NoC|, 20 ml 0TT, p¬ 7.4
Flow rates: $omp|e |ood|ng, 0.3 m|/m|n wosh ond e|ut|on, l m|/m|n |^Cl l.5 m|/m|n |Cfl
System: ^KT^xpress
SDS-PAGE
500
0
1000
1500
mAU
0 0 2 0 5 150 100
AC GF
ml
A
280
Large aggregates of
GST-hippocalcin
Dimers of GST-hippocalcin
150 140 130 120 110 ml
50
0
100
150
200
250
mAU
A
280
97
66
45
30
20
14
(M
r
× 10
3
)
GST-hippocalcin
L
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m
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o
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h

(
A
C
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E
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f
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s

o
f

d
i
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e
r

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F
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d

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s
l2
lncreos|ng the pur|ncot|on sco|e
Products featured: pGEX-2T, GSTrap FF, GSTPrep FF 16/10, ÄKTAexplorer, ExcelGel SDS Gradient 8–18,
Multiphor II Electrophoresis System, LMW-SDS Marker Kit
0nce o method íor the pur|ncot|on oí o torget prote|n hos been estob||shed, |t con be sco|ed up to produce |orger quont|t|es oí
torget prote|n íor íunct|ono| ond structuro| stud|es. The one-step pur|ncot|on method be|ow |||ustrotes o 26-ío|d sco|e-up. The mo|n
porometer |n th|s sco|e-up studg wos res|dence t|me |the per|od oí t|me the somp|e |s |n contoct w|th the chromotogrophg med|uml.
Res|dence t|me wos the some íor the C$Trop ff l m| ond 5 m| co|umns, but tw|ce os |ong íor the C$Tlrep¯ ff l6/l0 |20 m| co|umnl
due to the d|ííerence |n co|umn |ength ond d|ometer. The g|e|d wos determ|ned bg meosur|ng the obsorbonce ot 280 nm ond pur|tg
wos ossessed bg $0$-l^CL.
Columns: C$Trop ff l m|, C$Trop ff 5 m|, ond
C$Tlrep ff l6/l0 |20 m|l
Sample: C$T-0em^ |n E. coli extroct
Sample volumes: l0 m| |l m| co|umnl, 50 m|
|5 m| co|umnl, 200 m| |20 m| co|umnl
Binding buffer: l3$, p¬ 7.4
Elution buffer: 50 ml Tr|s-¬C|, l0 ml reduced
g|utoth|one p¬ 8.0
Flow rates: 0.5 m|/m|n ot somp|e |ood|ng, l m|/m|n
ot wosh|ng ond e|ut|on |C$Trop ff l m|l
2.5 m|/m|n ot somp|e |ood|ng, 5 m|/m|n ot wosh|ng
ond e|ut|on |C$Trop ff 5 m|l
5 m|/m|n ot somp|e |ood|ng, l0 m|/m|n ot wosh|ng
ond e|ut|on |C$Tlrep ff l6/l0l
System: ^KT^exp|orer l00
SDS-PAGE
Summary
^n |ncreosed co|umn vo|ume ond somp|e |ood |ed to o proport|ono||g |ncreosed g|e|d oí the e|uted C$T-togged prote|n |n the
sco|e-up protoco|s.
mAU
1200
1000
800
600
400
200
0
A
280
Elution buffer
100
80
60
40
20
0
%B
0
Wash
Elution
buffer
5.5 mg
10 20 30 ml
1400
1200
1000
800
600
400
200
0
A
280
100
80
60
40
20
0
%B
Wash
Elution
buffer
20.9 mg mAU
0 50 100 150 ml
Elution buffer
Elution
buffer
A
280
mAU
1400
1200
1000
800
600
400
200
0
Elution buffer
100
80
60
40
20
0
%B
Wash
111.0 mg
0 100 200 300 400 500 600 ml
GST-DemA
97
66
45
30
20
14
(M
r
× 10
3
)
L
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W

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m
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t

m
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d

f
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o
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s
F
l
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E
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d

f
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t

m
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s
GSTrap FF 1 ml GSTrap FF 5 ml GSTPrep FF 16/10
GSTrap FF 1 ml GSTrap FF 5 ml GSTPrep FF 16/10
Removo| oí |orge prote|n togs such os the C$T tog |s oíten necessorg íor torget prote|n chorocter|zot|on. The omount oí
proteose, temperoture, ond |ength oí |ncubot|on requ|red íor comp|ete d|gest|on vor|es occord|ng to the noture oí the
port|cu|or torget prote|n.
lre$c|ss|on lroteose |s on eínc|ent enzgme íor the spec|nc c|eovoge ond removo| oí C$T togs. lre$c|ss|on lroteose |s
bg |tse|í o togged prote|n oí C$T ond humon rh|nov|rus 3C proteose, hence |t |s eos||g removed írom the c|eoved torget
prote|n |o|ong w|th C$Tl us|ng C|utoth|one $ephorose oínn|tg med|um. $|nce lre$c|ss|on lroteose |s opt|mo||g oct|ve ot
4ºC, c|eovoge con be períormed ot |ow temperotures to enhonce the stob|||tg oí the torget prote|n.
Un||ke other proteoses, the recogn|t|on s|te oí lre$c|ss|on lroteose |s not o noturo||g occurr|ng sequence ond th|s coníers
o h|gh degree oí spec|nc|tg on the enzgme. C|eovoge oí C$T-togged prote|ns con o|so be períormed w|th other proteoses
thot recogn|ze d|ííerent c|eovoge s|tes. foctor ×o proteose ond Thromb|n proteose ore ser|ne proteoses w|th opt|mo|
c|eovoge períormonce ot room temperoture.
Tog c|eovoge enzgmes
Cleavage site
Glutathione
GST
Recombinant
protein
Sepharose
Cleavage enzymes
Factor Xa protease: l
r
28 000 - 30 000
Thrombin protease: l
r
37 000
PreScission Protease: l
r
46 000
G
S
T
-
t
a
g
g
e
d

p
r
o
t
e
i
n
s
l3
G
S
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-
t
a
g
g
e
d

p
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s
l4
0pt|m|zot|on oí tog c|eovoge
Products featured: GSTrap FF, PreScission Protease, ÄKTAxpress
^n opt|mo| cond|t|on íor c|eovoge wos eínc|ent|g |dent|ned bg screen|ng exper|ments w|th on-co|umn c|eovoge oí o C$T-togged
prote|n us|ng C$Trop ff ond lre$c|ss|on lroteose on on ^KT^xpress sgstem. lncubot|on t|me ond the rot|os oí proteose ond
togged prote|n were vor|ed. The percentoge oí c|eoved prote|n wos determ|ned bg |ntegrot|ng the oreo oí the the e|uted, c|eoved
prote|n ond d|v|d|ng th|s w|th the |ntegroted toto| peok oreo |e|uted c|eoved prote|n + unc|eoved prote|nl. The c|eovoge reoct|on
wos períormed ot 4ºC.
Summary
The opt|m|zed cond|t|ons |dent|í|ed were 20 U oí proteose/mg oí torget prote|n íor o toto| |ncubot|on t|me oí 8 h. Th|s opt|mo|
cond|t|on wos used íor íurther stud|es.
0
10
20
30
40
50
60
70
80
90
100
10 units PreScission/mg protein
20 units PreScission/mg protein
40 units PreScission/mg protein
2 4 6 8 10 12 14
Incubation time (h)
C
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a
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d

p
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(
%
)
0
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-
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d

p
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e
i
n
s
l5
lur|ncot|on ond
on-co|umn c|eovoge
Products featured: GSTrap FF, Thrombin protease, ÄKTAexplorer, ExcelGel SDS Gradient 8–18, Multiphor II
Electrophoresis System, LMW-SDS Marker Kit
C|eovoge oí the C$T oínn|tg tog con e|ther be períormed on-co|umn beíore e|ut|on or |n so|ut|on oíter e|ut|on oí the torget mo|ecu|e.
To demonstrote the eínc|encg oí on-co|umn c|eovoge |n conjunct|on w|th pur|ncot|on, o C$T-togged prote|n conto|n|ng the
recogn|t|on sequence íor Thromb|n proteose wos opp||ed to o C$Trop ff l m| co|umn. ^íter |ncubot|on w|th the proteose íor l6 h ot
room temperoture, the C$T-íree torget prote|n wos e|uted.
SDS-PAGE
Summary
Lííect|ve on-co|umn c|eovoge oí the C$T mo|etg w|th Thromb|n proteose con be |ntegroted w|th the C$T pur|í|cot|on process.
Column: C$Trop ff l m|
Sample: l0 m| oí c|or|ned cgtop|osm|c extroct írom E. coli express|ng o C$T-togged prote|n
Binding buffer: l3$, p¬ 7.3
Elution buffer: 50 ml Tr|s-¬C|, l0 ml reduced g|utoth|one, p¬ 8.0
Flow rate: l m|/m|n
System: ^KT^exp|orer l0

0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
5.0 10.0 15.0 min
Wash
Incubation
16 h
room temp.
Wash
A280
Target
protein
Free
GST
2.0 4.0 6.0 8.0 10.0 12.0 min
0
20
40
60
80
100
%
Elution
buffer
97
66
45
30
20
14
(M
r
× 10
3
)
L
M
W

m
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t

m
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a
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G
S
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-
t
a
g
g
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d

p
r
o
t
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i
n
,

1

m
l

c
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l
u
m
n
G
S
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-
t
a
g
g
e
d

p
r
o
t
e
i
n
,

5

m
l

c
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l
u
m
n
G
S
T
-
f
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e

t
a
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t

p
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t
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n
,

1
6

h

c
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a
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C
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a
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d

G
S
T

t
a
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T
h
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m
b
i
n

p
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o
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a
s
e

(
2
0

U
/
m
l
)
L
M
W

m
a
r
k
e
r
s
Thrombin
protease
cleavage,
GSTrap FF 1 ml
Target
protein
G
S
T
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a
g
g
e
d

p
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o
t
e
i
n
s
l6
^utomoted mu|t|step
pur|ncot|on ond tog removo|
Products featured: pGEX-6P, GSTrap HP, PreScission Protease, HiLoad 16/60 Superdex 75 pg, ÄKTAxpress,
ExcelGel SDS gradient 8–18, Multiphor II Electrophoresis System, LMW-SDS Marker Kit
C$T-togged prote|ns produced w|th o lre$c|ss|on lroteose c|eovoge s|te enob|e two-step pur|ncot|on w|th on-co|umn tog c|eovoge
|n the nrst oínn|tg step. ^ C$T-togged mode| prote|n wos pur|ned w|th ^KT^xpress us|ng on outomoted two-step pur|ncot|on method.
C|eovoge ond removo| oí the C$T tog wos |mp|emented |n the protoco|.
Summary
Tog c|eovoge ond removo| were |ncorporoted |nto the outomoted two-step pur|í|cot|on protoco| ond th|s produced o h|gh|g
pure ond c|eoved torget prote|n.
Columns: ^ínn|tg chromotogrophg |^Cl, C$Trop ¬l 5 m|,
Ce| n|trot|on |Cfl, ¬|Lood l6/60 $uperdex 75 pg
Sample: C$T-purα |l
r
6l 600l
Binding/cleavage buffer (AC): 50 ml Tr|s-¬C|, l50 ml NoC|, l ml L0T^,
l ml 0TT, p¬ 7.5
Elution buffer (AC): 50 ml Tr|s-¬C|, l0 ml reduced g|utoth|one, p¬ 8.0
Buffer (GF): 50 ml Tr|s-¬C|, l50 ml NoC|, p¬ 7.5
System: ^KT^xpress
SDS-PAGE
AC GF
0
500
1000
1500
2000
mAU
200 250 300
46 mg
Cleaved protein
Regeneration
A
280
ml
97
66
45
30
20
14
(M
r
× 10
3
)
L
M
W

m
a
r
k
e
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s
S
t
a
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t

m
a
t
e
r
i
a
l
F
l
o
w
t
h
r
o
u
g
h
P
u
r
i
f
i
e
d
,

c
l
e
a
v
e
d

G
S
T
-
p
u
r

U
n
c
l
e
a
v
e
d

G
S
T
-
p
u
r

pur
G
S
T
-
t
a
g
g
e
d

p
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o
t
e
i
n
s
l7
0etect|on oí
C$T-togged prote|ns
lrote|n |mmunodetect|on |s o detect|on method thot meosures o spec|nc ont|gen/ont|bodg reoct|on. C$T 96-we|| 0etect|on lodu|e
mokes |t poss|b|e to v|suo||ze ond quont|tote C$T-togged prote|ns írom o comp|ex prote|n somp|e. The wog o recomb|nont prote|n
ío|ds con somet|mes mosk some oí |ts b|nd|ng s|tes dur|ng ont|bodg detect|on oí C$T-togged prote|ns. The use oí on ont|-C$T po|gc|ono|
ont|bodg copob|e oí recogn|z|ng more thon one ep|tope on C$T-togged prote|ns greot|g enhonces the chonces oí detect|on.
4 5 6 7 8 9 10 11 12 1 2 3
Control Anti-luciferase detection
Columns
l-3 $er|o| d||ut|ons oí contro| C$T-|uc|íerose
4-l2 50 µ| oí c|or|ned |gsotes írom se|ected co|on|es
ldent|ncot|on oí c|ones
express|ng C$T-togged prote|n
Products featured: pGEX-6P-1, GST 96-well Detection Module
0nce the gene oí |nterest hos been c|oned |nto the pCL× express|on vector ond the host ce||s used íor the c|on|ng step hove been
tronsíormed, chem|co||g |nduc|b|e h|gh-|eve| express|on oí the torget prote|n |s poss|b|e. The next step |s to opt|m|ze C$T-togged
prote|n express|on ond o keg step |s the copob|||tg to screen |gsotes írom mong c|ones.
Cu|tures oí rondom|g se|ected E. coli co|on|es resu|t|ng írom o pCL×-6l-l/|uc|íerose gene c|on|ng exper|ment were grown, |nduced, ond
|gsed |n o 96-we|| p|ote. Coptured C$T-togged |uc|íerose detected w|th robb|t ont|-|uc|íerose, ont|-robb|t lgC/perox|dose conjugote
us|ng C$T 96-we|| 0etect|on lodu|e. Tl3 |3,3,5,5-tetromethg| benz|d|nel wos used os substrote ond the obsorbonce wos reod ot 450 nm.
Summary
f|ítg-three percent oí the c|ones tested expressed C$T-togged |uc|íerose.
l8
G
S
T
-
t
a
g
g
e
d

p
r
o
t
e
i
n
s
SDS-PAGE
97
66
45
30
20
14
(M
r
× 10
3
)
S
o
n
i
c
a
t
e
,

E
.

c
o
l
i

T
G
1
S
o
n
i
c
a
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e
,

K
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4
5

c
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l
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s

(
D
n
a
K

o
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x
p
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s
s
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)
S
o
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i
c
a
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e
,

E
.

c
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+

p
G
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X
-
5
X
-
1
S
o
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i
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a
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,

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c
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i

+

p
G
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X
-
5
X
-
L
u
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e
x
p
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s
s
i
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g

G
S
T
-
l
u
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f
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a
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S
o
n
i
c
a
t
e
,

E
.

c
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i

+

p
G
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X
-
4
T
-
E
7

e
x
p
r
e
s
s
i
n
g

G
S
T
-
E
7
P
u
r
i
f
i
e
d

G
S
T
P
r
e
s
t
a
i
n
e
d

M
W

m
a
r
k
e
r
vestern b|ot detect|on
Products featured: Anti-GST Antibody, Amersham Hybond ECL, Amersham Hyperfilm ECL, Amersham GST
Western Blotting Detection Kit
$omet|mes, E. coli ce||s mog hove d|íncu|tg overexpress|ng o port|cu|or torget prote|n. To test the eíncocg oí the express|on sgstem,
E. coli ce||s conto|n|ng o pCL×-5×-l vector w|thout on |nsert, were |nduced to overexpress the C$T mo|etg on|g. Two d|ííerent C$T-togged
prote|ns were then overexpressed |n E. coli ond detected bg the use oí ^nt|-C$T ^nt|bodg ond vestern b|ot us|ng LCL¯ detect|on.
HRP
Summary
^nt|-C$T ^nt|bodg prov|des o qu|ck ond conven|ent method íor detect|ng C$T-togged prote|ns.
L|ght
$econdorg ont|bodg
¬Rl conjugote
lr|morg ont|bodg
^nt|gen on membrone
LCL detect|on reogent
l9
0rder|ng |níormot|on
Product Quantity Code No.
Protein expression
pCL×-2T 25 µg 27-480l-0l
pCL×-2TK 25 µg 27-4587-0l
pCL×- 4T-l 25 µg 27-4580-0l
pCL×- 4T-2 25 µg 27-458l-0l
pCL×- 4T-3 25 µg 27-4583-0l
pCL×-3× 25 µg 27-4803-0l
pCL×lλT 5 µg 27-4805-0l
pCL×- 5×-l 25 µg 27-4584-0l
pCL×- 5×-2 25 µg 27-4585-0l
pCL×- 5×-3 25 µg 27-4586-0l
pCL×- 6l-l 25 µg 27-4597-0l
pCL×- 6l-2 25 µg 27-4598-0l
pCL×- 6l-3 25 µg 27-4599-0l
All vectors include L. co|| BL21
Purification
C$Trop ¬l 5 × l m| l7-528l-0l
l00 × l m|
¦
l7-528l-05
l × 5 m| l7-5282-0l
5 × 5 m| l7-5282-02
l00 × 5 m|
¦
l7-5282-05
C$Trop ff 2 × l m| l7-5l30-02
5 × l m| l7-5l30-0l
l00 × l m|
¦
l7-5l30-05
l × 5 m| l7-5l3l-0l
5 × 5 m| l7-5l3l-02
l00 × 5 m|
¦
l7-5l3l-05
C$Tlrep ff l6/l0 l × 20 m| l7-5234-0l
C$T lu|t|Trop ff 4 × 96-we|| n|ter
p|otes
28-4055-0l
C$Trop 43 5 × l m| 28-40l7-45
l00 × l m|
¦
28-40l7-46
l × 5 m| 28-40l7-47
5 × 5 m| 28-40l7-48
l00 × 5 m|
¦
28-40l7-49
C$T $p|nTrop
lur|ncot|on lodu|e
50 × 50 µ| 27-4570-03
C$T lu|t|Trop 43 4 × 96-we|| n|ter
p|otes
28-4055-00
C$T 0etect|on lodu|e 50 detect|ons 27-4590-0l
C$T 96-ve|| 0etect|on
lodu|e
5 p|otes 27-4592-0l
Detection
^nt|-C$T ^nt|bodg 0.5 m|,
50 detect|ons
27-4577-0l
^mershom LCL C$T vestern
3|ott|ng 0etect|on K|t
l k|t RlNl237
^mershom ¬gpern|m¯ LCL
|l8 x 24 cml
50 sheets 28-9068-36
^mershom ¬gbond¯ LCL
|20 × 20 cml
l0 sheets RlN20200
Tag cleavage
Thromb|n proteose 500 un|ts 27-0846-0l
foctor ×o proteose 400 un|ts 27-0849-0l
lre$c|ss|on lroteose 500 un|ts 27-0843-0l
Related products
C|utoth|one $ephorose ¬|gh
leríormonce
25 m| l7-5279-0l
l00 m| l7-5279-02
C|utoth|one $ephorose
4 fost f|ow
25 m| l7-5l32-0l
l00 m| l7-5l32-02
500 m| l7-5l32-03
C|utoth|one $ephorose 43 l0 m| l7-0756-0l
l00 m|
|íunct|on testedl
27-4574-0l
300 m| l7-0756-04
3u|k C$T lur|ncot|on lodu|e l k|t 27-4570-0l
Red|lock C$T lur|ncot|on
lodu|e
l k|t 27-4570-02
C|utoth|one $ephorose 43
|prepocked d|sposob|e
co|umnsl
2 x 2 m| l7-0757-0l
¬|Lood l6/60 $uperdex 30 pg l × l20 m| l7-ll39-0l
¬|Lood 26/60 $uperdex 30 pg l × 320 m| l7-ll40-0l
¬|Lood l6/60 $uperdex 75 pg l × l20 m| l7-l068-0l
¬|Lood 26/60 $uperdex 75 pg l × 320 m| l7-l070-0l
¬|Lood l6/60 $uperdex 200 pg l × l20 m| l7-l069-0l
¬|Lood 26/60 $uperdex 200 pg l × 320 m| l7-l07l-0l
Lxce|Ce|¯ $0$
Crod|ent 8-l8
6 80-l255-53
Llv-$0$ lorker K|t l0 v|o|s l7-0446-0l
Product Quantity Code No.

available by specific customer order
For more information on equipment for chromatography and/or electrophoresis, please visit www.gelifesciences.com
G
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¬|st|d|ne-togged prote|ns
¬|st|d|ne togs ore w|de|g used becouse theg ore smo|| ond rore|g |nteríere
w|th the íunct|on, oct|v|tg, or structure oí torget prote|ns. lmmob|||zed meto|
|on oínn|tg chromotogrophg |ll^Cl |s the most common method íor
pur|íg|ng h|st|d|ne-togged prote|ns. ll^C chromotogrophg med|o chorged
w|th d|vo|ent meto| |ons such os n|cke| se|ect|ve|g reto|n h|st|d|ne-togged
prote|ns ond o||ow íor the pur|ncot|on oí |nso|ub|e h|st|d|ne-togged prote|ns
írom |nc|us|on bod|es when denotur|ng cond|t|ons ore used. $uccessíu|
ll^C pur|ncot|on g|ves o h|gh g|e|d oí pure ond oct|ve torget prote|n.
¬owever, s|nce mong prote|ns hove |ntr|ns|c h|st|d|ne ond/or cgste|ne
om|no oc|d res|dues, other nonspec|nc prote|ns b|nd to the ll^C med|o
together w|th the torget prote|n. ln such coses, |t |s oíten necessorg to
opt|m|ze b|nd|ng, wosh, ond e|ut|on cond|t|ons bg vorg|ng the concentrot|on
oí |m|dozo|e |n these so|ut|ons. lncreos|ng the concentrot|on oí |m|dozo|e
|n the b|nd|ng ond wosh buííers genero||g decreoses nonspec|nc b|nd|ng,
whereos |ower concentrot|ons g|ve stronger oínn|tg |nteroct|on. The keg |s
nnd|ng the r|ght bo|once.
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lur|ncot|on oí h|st|d|ne-
togged prote|ns
0|ííerent N| $ephorose chromotogrophg med|o ore ovo||ob|e |n servero| íormots. The med|o vorg |n the|r períormonce porometers,
ond the d|ííerent íormots prov|de opt|ons íor sco|e ond conven|ence.
Conto|ns N| $ephorose ¬|gh leríormonce
Conto|ns N| $ephorose 6 fost f|ow
No
v||| gou use on outomoted pur|ncot|on sgstem such os ^KT^des|gn` \es
¬|slrep ff l6/l0
Crov|tg-fow co|umns
No
¬|s Crov|Trop
No
3otch
\es
¬|s Crov|Trop K|t
vhot ore gour requ|rements`
3uííers |nc|uded`
¬|s $p|nTrop
¬|s lu|t|Trop ff
N| $ephorose 6 fost f|ow
¬|s lu|t|Trop ¬l
96-we|| p|ote
$p|n co|umn
lrepocked co|umns
¬|sTrop ¬l
N| $ephorose ¬|gh leríormonce
\es No
$co|ob|||tg/h|gh fow rotes
lrepocked co|umns
\es
^mount oí h|st|d|ne-togged prote|n
> 200 mg < 200 mg
Ni Sepharose
¬|gh reso|ut|on
$gr|nge
3uííers |nc|uded`
¬|sTrop ff crude
No \es
¬|sTrop ff crude K|t
¬|sTrop ff
C|or|ned or unc|or|ned somp|e
Unc|or|ned C|or|ned
¬|s $p|nTrop K|t
3uííers |nc|uded`
\es No
2l
22
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^utomot|on oí h|gh-throughput
express|on screen|ng
Products featured: His MultiTrap HP
^utomoted ond reproduc|b|e protoco|s íor eínc|ent h|gh-throughput pur|ncot|on or express|on screen|ng oí togged prote|ns hove
become o keg step |n the seorch íor drug torgets. ^ two-port studg wos conducted to meosure the robustness oí on opt|m|zed,
outomoted protoco| períormed |n o vocuum on o ||qu|d hond||ng stot|on. f|rst, o chessboord studg wos períormed to meosure
poss|b|e cross-contom|not|on. ^ h|st|d|ne-togged prote|n somp|e wos opp||ed to everg second we|| ond the other we||s were
|eít emptg. ln the second port, the degree oí reproduc|b|||tg wos determ|ned bg the opp||cot|on oí s|x d|ííerent h|st|d|ne-togged
prote|ns rep||coted |n seven rows. The e|uted h|st|d|ne-togged prote|ns were ono|gzed bg $0$-l^CL.
Summary
^utomot|on methods |ncreose throughput ot eoch stoge ond enob|e o h|gh degree oí robustness w|th neg||g|b|e cross
contom|not|on ond o h|gh|g cons|stent we||-to-we|| períormonce.
96-well filter plate: ¬|s lu|t|Trop ¬l
Samples: Chessboord studg. Two E. coli somp|es, one
express|ng o |h|st|d|nel
6
-togged recomb|nont prote|n
|l
r
37 000l ond o somp|e where no recomb|nont prote|n
wos expressed.
Reproduc|b|||tg studg. $|x d|ííerent E. coli somp|es
express|ng d|ííerent s|zes oí recomb|nont prote|ns
|see $0$-l^CL be|owl. Two oí the s|x recomb|nont prote|ns
|2 ond 6l were not expressed.
Equilibration buffer: 20 ml Tr|s-¬C|, 500 ml NoC|,
20 ml |m|dozo|e, p¬ 7.4
Wash buffer: 20 ml Tr|s-¬C|, 500 ml NoC|,
25 ml |m|dozo|e, p¬ 7.4
Elution buffer: 20 ml Tr|s-¬C|, 500 ml NoC|,
500 ml |m|dozo|e, p¬ 7.4
Liquid handling station: ¬om||ton llCR0L^3¯ $T^R
Vacuum station: llCR0L^3 $T^R 3os|c vocuum $gstem |3v$l
100
75
50
37
25
20
15
10

1 2 3 4 5 6 1 2 3 4 5 6 1 2 3 4 5 6 1 2 3 4 5 6 1 2 3 4 5 6 1 2 3 4 5 6 1 2 3 4 5 6 1 2 3 4 5 6
M M M M
Clarified
bacterial
extracts
Elution,
row 4
Elution,
row 1
Elution,
row 2
Elution,
row 3
Elution,
row 5
Elution,
row 6
Elution,
row 7
(M
r
× 10
3
)
M B P B P B P B P B P B P B P B P B P B P B P B P B
B : Blank
P : Protein purification
(37 kDa)
B : Blank
P : Protein purification
(37 kDa)
B : Blank
P : Protein purification
(37 kDa)
150
100
75
50
37
25
20
15
10
(M
r
× 10
3
)
l = lrote|n pur|ncot|on |l
r
37 000l
3 = 3|onk
Acknowledgements: 3. Co||et, l. No|rc|erc-$ovoge ond T. vernet, Ro3|olo|/Loborotorg íor locromo|ecu|or Lng|neer|ng, lnst|tut de 3|o|og|e
$tructuro|e CL^-CNR$-UJf, Crenob|e, fronce
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23
0pt|m|z|ng pur|ncot|on
cond|t|ons
Products featured: His SpinTrap, ExcelGel SDS Gradient 8–18, Multiphor II Electrophoresis System,
LMW-SDS Marker Kit
The |m|dozo|e concentrot|on dur|ng b|nd|ng ond wosh|ng |s on |mportont íoctor thot oííects the nno| pur|tg ond g|e|d oí the torget
prote|n. Th|s wos demonstroted bg o ser|es oí exper|ments |n wh|ch o h|st|d|ne-togged prote|n, ^l3 7-|¬|sl
6
, |l
r
28 000l, wos pur|ned
on ¬|s $p|nTrop us|ng 5, 50, l00, or 200 ml |m|dozo|e concentrot|ons |n the somp|e ond b|nd|ng buííers.
97
66
45
30
20
14
(M
r
× 10
3
)
APB 7-(His)
6
L
M
W

m
a
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k
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s
S
t
a
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t

m
a
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e
r
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l
,

d
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e
d

1
:
1
0
5

m
M

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d
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g

b
i
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d
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g
,

d
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d

1
:
2
5
0

m
M

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d
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g

b
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d
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,

d
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d

1
:
2
1
0
0

m
M

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a
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d
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b
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d
i
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g
,

d
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d

1
:
2
2
0
0

m
M

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a
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d
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g

b
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d
i
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g
,

d
i
l
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e
d

1
:
2
Eluted pool
Column: ¬|s $p|nTrop
Sample: 600 µ| E.coli 3L-2l |gsote conto|n|ng 400 µg
^l3 7-|¬|sl
6
, l
r
28 000
Binding/wash buffer: 20 ml phosphote, 500 ml NoC|,
5 to 200 ml |m|dozo|e, p¬ 7.4
Elution buffer: 20 ml phosphote, 500 ml NoC|,
500 ml |m|dozo|e, p¬ 7.4
Summary
ln th|s opp||cot|on, 50 ml |m|dozo|e prevented the b|nd|ng oí most contom|nonts ond |mproved pur|tg. ^dd|ng more |m|dozo|e to
the somp|e ond b|nd|ng buííers |ed to o morg|no| |ncreose |n pur|tg but |ower prote|n g|e|d.
SDS-PAGE
24
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lonuo| grov|tg pur|ncot|on
Summary
3oth $0$-l^CL ond vestern b|ot ono|gses oí the e|uted íroct|ons showed three mojor bonds |nd|cot|ng thot h|st|d|ne togs
were present. The top bond |s the torgeted íu||-|ength prote|n ond the bonds be|ow represent truncoted íorms oí the h|st|d|ne-
togged torget prote|n.
Products featured: His GraviTrap Kit, PhastSystem, PhastGel Gradient 10–15, Amersham Hybond ECL,
Anti-His Antibody, ECL Mouse IgG, HRP-linked Whole Ab, Amersham High-Range Rainbow Molecular
Weight Markers
The grov|tg pur|ncot|on method con be eííect|ve when gou need to pur|íg |orge somp|es |n the obsence oí o chromotogrophg
sgstem. 0|rect pur|ncot|on w|thout pr|or c|or|ncot|on oí the bocter|o| ce|| |gsotes con be occomp||shed bg us|ng ¬|s Crov|Trop¯
co|umns. ln th|s exomp|e, o h|gh mo|ecu|or we|ght |h|st|d|nel
l0
-togged prote|n wos pur|ned |n 25 m|n írom 20 m| oí c|or|ned E. coli
Jll09 |gsote conto|n|ng TR×-l450-|¬|sl
l0
|l
r
~ l30 000l. The e|uted íroct|ons were ono|gzed bg $0$-l^CL ond vestern b|ott|ng.
H
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1
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0
F
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,

d
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d

1
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0
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1
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2
N
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a
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c
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(
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d

J
M
1
0
9
)

220
97
66
45
20
14
30
(M
r
× 10
3
)
H
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h

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a
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w
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t

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1
:
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0
F
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h
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,

d
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d

1
:
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0
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1
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2
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(
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1
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9
)
|¬|sl
l0
-TR×-l450
Western blot
Column: ¬|s Crov|Trop
Sample: 20 m| c|or|ned E.coli Jll09 |gsote conto|n|ng |¬|sl
l0
-TR×-l450 |l
r
~l30 000l
Binding/wash buffer: 20 ml phosphote, 500 ml NoC|, 40 ml |m|dozo|e, p¬ 7.4
Elution buffer: 20 ml phosphote, 500 ml NoC|, 500 ml |m|dozo|e, p¬ 7.4
SDS-PAGE
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p
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s
25
lonuo| pur|ncot|on us|ng
o sgr|nge
Products featured: HisTrap FF crude Kit, ExcelGel SDS Gradient 8–18, Multiphor II Electrophoresis System,
LMW-SDS Marker Kit
0|rect |ood|ng oí unc|or|ned ce|| |gsotes decreoses the toto| pur|ncot|on t|me ond |ncreoses the chonces oí pur|íg|ng sens|t|ve torget
prote|ns w|thout |os|ng oct|v|tg. ln the obsence oí o chromotogrophg sgstem, h|st|d|ne-togged prote|ns írom unc|or|ned ce|| |gsotes con
be pur|ned |n o motter oí m|nutes us|ng o co|umn ond sgr|nge. ln th|s exomp|e, conven|ent ond s|mp|e pur|ncot|on oí o h|st|d|ne-togged
mo|tose b|nd|ng prote|n írom on unc|or|ned somp|e wos períormed us|ng o sgr|nge ond the buííers |nc|uded |n ¬|sTrop¯ ff crude K|t.
Summary
l3l-|¬|sl
6
wos qu|ck|g |so|oted írom on unc|or|ned somp|e us|ng o sgr|nge.
0
30 25 20 15
Wash 1
Wash 2
Wash 3
Elution 1
10 5 0
0
0.5
1.0
1.5
2.0
2.5
3.0
100
200
300
400
500
Imidazole
concentration
(mM)
Elution volume (ml)
A
280
Elution 2
Elution 3
(M
r
× 10
3
)
97
66
45
30
20
14
MBP-(His)
6
L
M
W

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5

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,

1
0

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0

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,

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0

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0

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0

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3
,

5
0
0

m
M

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Column: ¬|sTrop ff crude l m|
Sample: Unc|or|ned E. coli extroct conto|n|ng
l3l-|¬|sl
6
, l
r
43 000
Flow rate: ^pprox. l m|/m|n
Buffers: lnc|uded |n the ¬|sTrop ff crude K|t
Fraction size: l m|
Equipment: lonuo| pur|ncot|on us|ng o sgr|nge
SDS-PAGE
l m|/íroct|on
26
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$|mp|e one-step pur|ncot|on
Summary
Líí|c|ent pur|í|cot|on us|ng step e|ut|on wos och|eved |n 35 m|n.
Products featured: HisTrap HP, ÄKTAprime plus
¬|gh|g pure h|st|d|ne-togged prote|ns con be obto|ned |n o s|ng|e pur|ncot|on step w|th o chromotogrophg sgstem. ln the exomp|e
shown, o step-grod|ent e|ut|on wos used to eínc|ent|g pur|íg o h|st|d|ne-togged prote|n. The pur|ncot|on process wos s|mp||ned bg
the use oí opt|m|zed protoco|s on on ^KT^pr|me¯ p|us.
Sample: C|or|ned homogenote oí E. coli express|ng h|st|d|ne-togged prote|n
Column: ¬|sTrop ¬l l m|
Binding buffer: 20 ml phosphote, 0.5 l NoC|, 20 ml |m|dozo|e, p¬ 7.4
Elution buffer: 20 ml phosphote, 0.5 l NoC|, 0.5 l |m|dozo|e, p¬ 7.4
System: ^KT^pr|me p|us
5 10 15 20 25 30 35 min
2.5
2.0
1.5
1.0
0.5
0
100
80
60
40
20
0
A
280
Elution
Buffer %
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27
lur|ncot|on oí o h|st|d|ne-togged
prote|n expressed |n Pichia pastoris
Products featured: HisTrap FF crude, ÄKTAexplorer, ExcelGel SDS Gradient 8–18, Multiphor II
Electrophoresis System, LMW-SDS Marker Kit
Lven |n coses where the torget h|st|d|ne-togged prote|n |s expressed ot verg |ow |eve|s, |t |s poss|b|e to obto|n re|ot|ve|g |orge
quont|t|es oí pure prote|n becouse the presence oí the h|st|d|ne tog eííect|ve|g enr|ches the torget prote|n on the oínn|tg co|umn.
^n unc|or|ned |gsote oí o h|st|d|ne-togged prote|n expressed |n Pichia pastoris wos |ooded d|rect|g onto o co|umn. The prote|n wos
e|uted us|ng o ||neor grod|ent.
Summary
^ h|gh|g pure torget prote|n wos obto|ned írom on unc|or|ned geost |gsote us|ng grod|ent e|ut|on.
Column: ¬|sTrop ff crude 5 m|
Sample: l30 m| oí unc|or|ned |gsote oí \NR064c |Saccharo-
myces cerevisiae hgdro|osel expressed |n P. pastoris
Binding buffer: 20 ml sod|um phosphote, 500 ml NoC|,
75 ml |m|dozo|e, p¬ 7.4
Elution buffer: 20 ml sod|um phosphote, 500 ml NoC|,
500 ml |m|dozo|e, 3 ml KC|, p¬ 7.4
Flow rate: 5 m|/m|n
System: ^KT^exp|orer l00
mAU
5000
4000
3000
2000
1000
0
100
50
0
0 100 200 300 400 ml
Elution
Buffer %
A
280
(M
r
× 10
3
)
97
66
45
30
20
14
(His)
6
-YNR064c
L
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SDS-PAGE
28
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0ne-step pur|ncot|on comb|ned
w|th on-co|umn reío|d|ng
Products featured: HisTrap HP, ÄKTAexplorer, Biacore System
$ome h|st|d|ne-togged prote|ns ore expressed os |nc|us|on bod|es |n E. coli. lnc|us|on bod|es ore |nso|ub|e oggregotes oí m|sío|ded
prote|ns |ock|ng b|o|og|co| oct|v|tg. The prob|em con be so|ved bg us|ng o s|mp|e, but eínc|ent comb|ned pur|ncot|on ond on-co|umn
reío|d|ng process to produce pure ond oct|ve prote|n. ^ h|st|d|ne-togged scfv 57l ont|bodg írogment expressed os |nc|us|on bod|es
|n E. coli wos so|ub|||zed |n guon|d|ne hgdroch|or|de ond opp||ed to o ¬|sTrop ¬l co|umn. Contom|nonts were removed ío||owed bg
on-co|umn reío|d|ng v|o buííer exchonge to o nondenotur|ng buííer. ^ b|nd|ng ossog between o pept|de der|ved írom the tobocco moso|c
v|rus ond the e|uote |reío|ded h|st|d|ne-togged prote|nl wos períormed us|ng suríoce p|osmon resononce |$lRl on o 3|ocore¯ sgstem.
Summary
Us|ng o comb|ned pur|í|cot|on ond on-co|umn reío|d|ng procedure produced l4% oí oct|ve, reío|ded prote|n.
500
400
300
200
100
0
91.5 92 93
pool
94 95 ml 92.5 93.5 94.5
Elution buffer %
100
80
60
40
20
0
500
400
300
200
100
0
0 20 40 60 80 ml
Elution
buffer %
mAU
100
80
60
40
20
0
1
4
2
3 5
Pooled fraction from the purification of refolded
scFv 57P antibody fragment
100
A
280
A
280
mAU
Sample: l0 m| oí so|ub|||zed h|st|d|ne-togged s|ng|e cho|n fv ont|bodg írogment |fob 57l
Column: ¬|sTrop ¬l l m|
Solubilizing buffer: 20 ml Tr|s-¬C|, 6 l Cuo-¬C|, l ml 0TL, l ml No
2
-L0T^, 0.l ml leíob|oc¯, p¬ 7.5
Denatured binding buffer: 20 ml Tr|s-¬C|, 5 ml |m|dozo|e, 0.5 l NoC|, 8 l ureo, l ml 0TL, 0.l ml leíob|oc, p¬ 7.5
Refolding buffer: 20 ml Tr|s-¬C|, 5 ml |m|dozo|e, 0.5 l NoC|, 0.5 l org|n|ne-¬C|, l ml reduced g|utoth|one |C$¬l, l ml ox|d|zed g|utoth|one |C$$Cl, p¬ 7.5
Native binding buffer: 20 ml Tr|s-¬C|, l0 ml |m|dozo|e, 0.5 l NoC|, p¬ 7.5
Native elution buffer: 20 ml Tr|s-¬C|, 500 ml |m|dozo|e, 0.5 l NoC|, p¬ 7.5
Flow rate: l m|/m|n
System: ^KT^exp|orer l0
l. $omp|e opp||cot|on
2. vosh
3. $tort reío|d|ng
4. Lqu|||brot|on oí co|umn w|th not|ve b|nd|ng buííer
5. $tort e|ut|on
5000
0
10 000
15 000
20 000
-5000
R
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R
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)
Reference cell
Sample cell
Response difference after subtraction of reference cell
200 400 600 800 1000 1200 1400 1600
s
SPR sensogram
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29
Summary
The eínc|ent, íour-step pur|ncot|on protoco| eííect|ve|g removed the h|st|d|ne tog írom the prote|n ond g|e|ded ll mg oí o h|gh|g
pure ^lCl040.
Unottended íour-step pur|ncot|on
ond outomot|c tog removo|
Products featured: HisTrap HP, HiPrep 26/10 Desalting, RESOURCE Q, HiLoad 16/60 Superdex 75 pg,
ÄKTAxpress, ExcelGel SDS Gradient 8–18, Multiphor II Electrophoresis System, LMW-SDS Marker Kit
^KT^xpress enob|es opt|m|zot|on oí c|eovoge cond|t|ons oí the h|st|d|ne tog ond outomotes the tosk oí mu|t|step pur|ncot|on.
0pt|m|zot|on oí ^cTLv¯ proteose c|eovoge cond|t|ons íor h|st|d|ne-togged ^lCl040 wos determ|ned us|ng ^KT^xpress. ^ íour-step
pur|ncot|on protoco| |nc|ud|ng on-co|umn tog c|eovoge us|ng the opt|m|zed c|eovoge cond|t|ons obto|ned wos then períormed to
och|eve h|gh omounts oí pure ^lCl040.
0
500
1000
1500
mAU
1100 1200 1300 1400 1500
B10
11 mg
Cleaved protein
Regeneration
A
280
ml
Elution
buffer %
100
80
60
40
20
0
AC DS IEX GF
97
66
45
30
20
14
(M
r
× 10
3
)
Cleaved APC1040 (Mr 36 400)
Uncleaved APC1040 (Mr 38 900)
L
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0

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4
0
Sample: ^lCl040-|¬|sl
6
|n E. coli |gsote
Columns: AC: ¬|sTrop ¬l 5 m|
DS: ¬|lrep¯ 26/l0 0eso|t|ng
IEX: RL$0URCL¯ Ç, 6 m|
GF: ¬|Lood l6/60 $uperdex 75 pg
Cleavage conditions: 200 un|ts oí ^cTLv proteose/mg prote|n,
8 h |ncubot|on t|me ot room temperoture
AC binding buffer: 50 ml Tr|s-¬C|, 500 ml NoC|, 20 ml |m|dozo|e, p¬ 7.5
AC cleavage buffer: 50 ml Tr|s-¬C|, 500 ml NoC|, 50 ml |m|dozo|e, p¬ 7.5
AC elution buffer: 50 ml Tr|s-¬C|, 500 ml NoC|, 500 ml |m|dozo|e, p¬ 7.5
DS and IEX binding buffer: 50 ml Tr|s-¬C|, p¬ 8.0
IEX elution buffer: 50 ml Tr|s-¬C|, l l NoC|, p¬ 8.0
GF buffer: 50 ml Tr|s-¬C|, l50 ml NoC|, p¬ 7.5
30
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lncreos|ng the pur|ncot|on sco|e
Products featured: HisTrap FF columns, HisPrep FF 16/10, ÄKTAexplorer, ExcelGel SDS Gradient 8–18,
Multiphor II Electrophoresis System, LMW-SDS Marker Kit
To obto|n suínc|ent moter|o| íor chorocter|zot|on stud|es, o sco|e-up exper|ment oí o h|st|d|ne-togged mo|tose b|nd|ng prote|n
|l3l-¦¬|s¦
6
l wos períormed. The some prote|n |ood ond ||neor fow rotes were used on o|| three co|umns. Recoverg ond pur|tg oí the
e|uted moter|o| wos determ|ned ond compored íor o|| three runs.
Summary
The three d|ííerent sco|e-up pur|í|cot|ons produced s|m||or resu|ts |n terms oí pur|tg ond recoverg.
Columns: ¬|sTrop ff l m|, ¬|sTrop ff 5 m|,
¬|slrep¯ ff l6/l0 |20 m|l
Sample: l3l-|¬|sl
6
, |n E. coli extroct
Sample volumes: 5.3 m| |l m| co|umnl,
26.5 |5 m| co|umnl, l06 m| |20 m| co|umnl
Binding buffer: 20 ml sod|um phosphote,
25 ml |m|dozo|e, 500 ml NoC|, p¬ 7.4
Elution buffer: 20 ml sod|um phosphote,
500 ml |m|dozo|e, 500 ml NoC|, p¬ 7.4
Flow rates: ¬|sTrop ff l m|. l m|/m|n,
¬|sTrop ff 5 m|. 5 m|/m|n,
¬|slrep ff l6/l0. 5 m|/m|n
System: ^KT^exp|orer l00
SDS-PAGE
A
280
0.0 50 100 150 ml
mAU
4000
3500
3000
2500
2000
1500
1000
500
0
33 mg
100
0
50
Elution
buffer %
HisTrap FF 1 ml HisTrap FF 5 ml HisPrep FF 16/10
0.0 5.0 10.0 15.0 20.0 25.0 30.0 40.0 ml
mAU
4000
3500
3000
2500
2000
1500
1000
500
0
6.2 mg
A
280
100
0
50
Elution
buffer %
100
0
50
Elution
buffer %
A
280
mAU
4000
3500
3000
2500
2000
1500
1000
500
0
0 100 200 300 500 400 600 ml
149 mg
700
97
66
45
30
20
14
(M
r
× 10
3
)
MBP-(His)
6
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3l
0rder|ng |níormot|on
Product Quantity Code No.
Purification
¬|sTrop ¬l 5 × l m| l7-5247-0l
l00 × l m|
¦
l7-5247-05
l × 5 m| l7-5248-0l
5 × 5 m| l7-5248-02
l00 × 5 m|
¦
l7-5248-05
¬|s lu|t|Trop ¬l 4 × 96-we|| n|ter
p|otes
28-4009-89
¬|s $p|nTrop 50 × l00 µ| 28-40l3-53
¬|s $p|nTrop K|t 50 x l00 µ|,
buííers
28-932l-7l
¬|sTrop ff 5 × l m| l7-53l9-0l
l00 × l m|
¦
l7-53l9-02
5 × 5 m| l7-5255-0l
l00 × 5 m|
¦
l7-5255-02
¬|sTrop ff crude 5 × l m| ll-0004-58
l00 × l m|
¦
ll-0004-59
5 × 5 m| l7-5286-0l
l00 × 5 m|
¦
l7-5286-02
¬|sTrop ff crude K|t 3 × l m|, buííers 28-40l4-77
¬|slrep ff l6/l0 l × 20 m| l7-5256-0l
¬|s lu|t|Trop ff 4 × 96-we|| n|ter
p|otes
28-4009-90
¬|s Crov|Trop l0 × l m| ll-0033-99
¬|s Crov|Trop K|t 20 × l m|, buííers 28-40l3-5l
Detection
^nt|-¬|s ont|bodg l70 µ| 27-47l0-0l
LCL louse lgC ¬Rl-||nked
vho|e ^b
l00 µ| N^93l-l00 µ|
^mershom ¬gbond LCL
|20 × 20 cml
l0 sheets RlN20200
^mershom ¬gpern|m LCL
|l8 × 24 cml
50 sheets 28-9068-36
Related products
¬|s 3uííer K|t l ll-0034-00
N| $ephorose ¬|gh
leríormonce
25 m| l7-5268-0l
l00 m| l7-5268-02
N| $ephorose 6 fost f|ow 5 m| l7-53l8-06
25 m| l7-53l8-0l
l00 m| l7-53l8-02
500 m| l7-53l8-03
¬|Lood l6/60 $uperdex 30 pg l × l20 m| l7-ll39-0l
¬|Lood 26/60 $uperdex 30 pg l × 320 m| l7-ll40-0l
¬|Lood l6/60 $uperdex 75 pg l × l20 m| l7-l068-0l
¬|Lood 26/60 $uperdex 75 pg l × 320 m| l7-l070-0l
¬|Lood l6/60 $uperdex 200 pg l × l20 m| l7-l069-0l
¬|Lood 26/60 $uperdex 200 pg l × 320 m| l7-l07l-0l
¬|lrep 26/l0 0eso|t|ng l × 53 m| l7-5087-0l
4 × 53 m| l7-5087-02
RL$0URCL Ç 6 m| l l7-ll79-0l
Lxce|Ce| $0$
Crod|ent 8-l8
6 80-l255-53
lhostCe|¯ Crod|ent l0-l5 l0 l7-0540-0l
Llv-$0$ lorker K|t l0 v|o|s l7-0446-0l
^mershom ¬|gh-Ronge
Ro|nbow lo|ecu|or ve|ght
lorkers
250 µ| RlN756L
Product Quantity Code No.
For more information on Biacore systems and chromatography and/or electrophoresis systems, please visit www.gelifesciences.com
¦
available by specific customer order
32
Strep-tog ll prote|ns
Strep-tog ll |s o smo|| tog oí on|g e|ght om|no oc|ds |Trp-$er-¬|s-lro-C|n-
lhe-C|u-Lgsl w|th o mo|ecu|or we|ght oí just l000. The smo|| s|ze oí the
tog |s verg benenc|o|, s|nce |n most coses |t does not |nteríere w|th structuro|
ond íunct|ono| stud|es ond, thereíore, does not hove to be removed.
Strep-tog ll b|nds spec|nco||g to Strep-Toct|n¯ ||gond |mmob|||zed on o
$ephorose bose motr|x to g|e|d pure torget prote|n. The b|nd|ng oínn|tg
oí the Strep-tog ll to the |mmob|||zed ||gond |s neor|g l00-ío|d greoter
thon to streptov|d|n, mok|ng $trepToct|n $ephorose ¬|gh leríormonce
su|tob|e íor pur|íg|ng Strep-tog ll prote|ns. lur|ncot|ons ore run under
phgs|o|og|co| cond|t|ons, ond m||d e|ut|on w|th desth|ob|ot|n preserves
the oct|v|tg oí the torget prote|n.
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$|mp|e two-step pur|ncot|on
Products featured: StrepTrap HP, HiLoad 16/60 Superdex 75 pg, ÄKTAprime plus, ExcelGel SDS Gradient
8–18, Multiphor II Electrophoresis System, LMW-SDS Marker Kit
¬|gh|g pure Strep-tog ll prote|ns con be obto|ned w|th o s|mp|e chromotogrophg sgstem such os ^KT^pr|me p|us. |¬|sl
6
-mCherrg-
Strep-tog ll |l
r
~3l 000l wos pur|ned írom on E. coli |gsote us|ng o two-step protoco| |nvo|v|ng oínn|tg chromotogrophg w|th o
$trepTrop¯ ¬l l m| co|umn ío||owed bg ge| n|trot|on.
AC step:
Column: $trepTrop ¬l l m|
Sample: l0 m| oí E. coli c|or|ned |gsote
conto|n|ng |¬|sl
6
-mCherrg-Strep-tog ll
Flow rate: l m|/m|n
Binding buffer: l00 ml Tr|s-¬C|, l50 ml NoC|,
l ml L0T^, p¬ 8.0
Elution buffer: 2.5 ml desth|ob|ot|n |n
b|nd|ng buííer
GF step:
Column: ¬|Lood l6/60 $uperdex 75 pg
Sample: L|uted poo| |2 m|l írom $trepTrop ¬l l m|
Flow rate: l m|/m|n
Buffer: l3$ buííer, p¬ 7.4
System: ^KT^pr|me p|us
2000
1500
0
A
280
mAU
1000
500
0 10 20 30 40 ml
40
50
30
60
70
0
20
10
0 10 20 30 40 50 60 ml
A
280
mAU
Summary
¬|gh|g pure Strep-tog ll recomb|nont prote|n wos obto|ned w|th th|s s|mp|e two-step pur|í|cot|on protoco|. The two
contom|nonts oí obout l
r
l0 000 ond 2l 000, respect|ve|g mog be coused bg írogmentot|on oí the torget prote|n dur|ng
$0$-l^CL ono|gs|s.
(Histidine)
6
tag Strep-tag II mCherry Full-length target protein
(Histidine)
6
tag
Strep-tag II
mCherry fragment
mCherry fragment
M
r
= 30 000
PreScission MYG (85-87) TEV
PreScission
MYG (85-87) TEV
M
r
9558
M
r
20 741
L
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Strep1rcp HP
£luted frcctions,
HiLocd 16l60
Superdex 75 pg
97
66
45
30
20
14
(M
r
× 10
3
)
SDS-PAGE
AC step GF step
34
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Unottended pur|ncot|on oí o
prote|n expressed |n |nsect ce||s
Products featured: StrepTrap HP, ÄKTAxpress
|¬|sl
6
-Strep-tog ll-prote|n |l
r
l5 400l wos expressed |n |nsect ce||s írom o 3ocu|ov|rus express|on vector ond pur|ned. The h|gh
spec|nc|tg oí the Strep-tog ll to the Strep-Toct|n ||gond wos ut|||zed |n o one-step pur|ncot|on procedure us|ng $trepTrop ¬l co|umn
to obto|n h|gh|g pure torget prote|n.
Column: $trepTrop ¬l 5 m|
Sample: l75 m| oí |nsect ce|| |gsote conto|n|ng
|¬|sl
6
-Strep-tog ll-prote|n |l
r
~l5 400l
Binding buffer: l00 ml Tr|s-¬C|, l50 ml NoC|, l ml L0T^, p¬ 8.0
Elution buffer: 2.5 ml desth|ob|ot|n |n b|nd|ng buííer
Flow rates: 5 m|/m|n |l m|/m|n dur|ng somp|e opp||cot|onl
System: ^KT^xpress
188
98
62
49
38
28
17
14
60
30
(M
r
× 10
3
)
(His)
6
-Strep-
tag II-protein
M
a
r
k
e
r
s
L
y
s
a
t
e
F
l
o
w
t
h
r
o
u
g
h
E
l
u
t
e
d

t
a
r
g
e
t

p
r
o
t
e
i
n

p
o
o
l
Summary
Th|s s|mp|e, s|ng|e-step process produced 3.7 mg oí h|gh|g pure prote|n.
SDS-PAGE
Acknowledgements: l. N||sson, R. $vensson ond L. ¬o|mgren, 3|ov|trum, $tockho|m, $weden
35
S
t
r
e
p
-
t
a
g

I
I

p
r
o
t
e
i
n
s
lncreos|ng the pur|ncot|on sco|e
Products featured: StrepTrap HP columns, XK 26/20 column, StrepTactin Sepharose High Performance,
ÄKTAexplorer
$co|e-up oí prote|ns con be och|eved bg |ncreos|ng the bed vo|ume wh||e keep|ng the res|dence t|me constont. Th|s opprooch
mo|nto|ns chromotogroph|c períormonce dur|ng sco|e-up. The prote|n used wos o fuorescent prote|n, |¬|sl
6
-mCherrg-Strep-tog ll, |n
E. coli |gsote, wh|ch con be detected ot 587 nm os we|| os 280 nm. lur|ncot|on on o $trepTrop ¬l l m| co|umn wos nrst períormed
ond then sco|ed up to the 5 m| co|umn ío||owed bg íurther sco|e-up to o 29 m| ×K 26/20 co|umn pocked w|th $trepToct|n $ephorose
¬|gh leríormonce. The prote|n |ood wos |ncreosed nve-ío|d |n eoch sco|e-up step.
Summary
The co|umns gove comporob|e resu|ts, coní|rm|ng the eose ond reproduc|b|||tg oí sco||ng up the pur|í|cot|on írom $trepTrop ¬l
co|umns to o |orger ×K 26/20 co|umn pocked w|th $trepToct|n $ephorose ¬|gh leríormonce.
0
1000
2000
3000
A280
mAU
0
1000
2000
3000
A587
mAU
0.0 10.0 20.0 ml
= A280
= A587
= Elution buffer, %B
Columns: $trepTrop ¬l l m|, $trepTrop ¬l 5 m|, $trepToct|n $ephorose
¬|gh leríormonce pocked |n ×K 26/20, 29 m|, bed he|ght 5.5 cm
Sample: |¬|sl
6
-mCherrg-Strep-tog ll |l
r
~3l 000l, |n E. coli |gsote
Sample volumes: 4.2 m| |$trepTrop ¬l l m|l, 2l m| |$trepTrop ¬l 5 m|l,
l05 m| |×K 26/20 co|umnl
Regeneration: 3 co|umn vo|umes |Cvl d|st|||ed woter, 3 Cv 0.5 l No0¬,
3 Cv d|st|||ed woter
Binding buffer: l00 ml Tr|s-¬C|, l50 ml NoC|, l ml L0T^, p¬ 8.0
Elution buffer: 2.5 ml desth|ob|ot|n |n b|nd|ng buííer
Flow rates: $trepTrop ¬l l m|. l.0 m|/m|n |0.5 m|/m|n dur|ng somp|e
|ood|ng ond regenerot|on w|th 0.5 l No0¬l.
$trepTrop ¬l 5 m|. 5.0 m|/m|n |2.5 m|/m|n dur|ng somp|e |ood|ng ond
regenerot|on w|th 0.5 l No0¬l.
×K 26/20 co|umn. l3 m|/m|n |6.5 m|/m|n dur|ng regenerot|on w|th
0.5 l No0¬l
System: ^KT^exp|orer
A280
mAU
A587
mAU
0
1000
2000
3000
0
1000
2000
3000
0 50 100 150 ml
A280
mAU
A587
mAU
0
1000
2000
3000
0
1000
2000
3000
0 200 400 600 700 ml
StrepTrap HP 1 ml StrepTrap HP 5 ml StrepTactin Sepharose
High Performance XK 26/20
36
S
t
r
e
p
-
t
a
g

I
I

p
r
o
t
e
i
n
s
0rder|ng |níormot|on
Purification
$trepTrop ¬l 5 × l m| 28-9075-46
l × 5 m| 28-9075-47
5 × 5 m| 28-9075-48
$trepToct|n $ephorose ¬|gh
leríormonce
l0 m| 28-9355-99
50 m| 28-9356-00
Related products
¬|Lood l6/60 $uperdex 30 pg l × l20 m| l7-ll39-0l
¬|Lood 26/60 $uperdex 30 pg l × 320 m| l7-ll40-0l
¬|Lood l6/60 $uperdex 75 pg l × l20 m| l7-l068-0l
¬|Lood 26/60 $uperdex 75 pg l × 320 m| l7-l070-0l
¬|Lood l6/60 $uperdex 200 pg l × l20 m| l7-l069-0l
¬|Lood 26/60 $uperdex 200 pg l × 320 m| l7-l07l-0l
¬|lrep 26/l0 0eso|t|ng l × 53 m| l7-5087-0l
4 × 53 m| l7-5087-02
Lxce|Ce| $0$ Crod|ent 8-l8 6 80-l255-53
Llv-$0$ lorker K|t l0 v|o|s l7-0446-0l
Product Quantity Code No.
For more information on chromatography and/or electrophoresis systems, please visit www.gelifesciences.com
37
38
l3l-togged prote|ns
lo|tose b|nd|ng prote|n |l3ll |s o useíu| oínn|tg tog thot con |ncreose the
express|on |eve| ond so|ub|||tg oí the resu|t|ng togged prote|n. The l3l tog
o|so promotes proper ío|d|ng oí the ottoched prote|n. $|nce l3l |ncreoses
so|ub|||tg, the tog |s port|cu|or|g useíu| íor recomb|nont prote|ns thot
occumu|ote |n on |nso|ub|e íorm ||nc|us|on bod|esl.
^ínn|tg pur|ncot|on tokes p|oce under phgs|o|og|co| cond|t|ons ond m||d
e|ut|on |s períormed us|ng mo|tose. The m||d e|ut|on preserves the oct|v|tg
oí the l3l-togged prote|n.
39
M
B
P
-
t
a
g
g
e
d

p
r
o
t
e
i
n
s
$|mp|e two-step pur|ncot|on
Products featured: MBPTrap HP, HiLoad 16/60 Superdex 200 pg, ÄKTAprime plus, ExcelGel SDS Gradient 8–18,
Multiphor II Electrophoresis System, LMW-SDS Marker Kit
l3l¯2-poromgos|n-δ-$o| wos pur|ned |n two steps w|th on l3lTrop¯ ¬l l m| co|umn, ío||owed bg ge| n|trot|on. The pur|ncot|on
process wos s|mp||ned w|th preprogrommed methods ond opt|m|zed protoco|s on on ^KT^pr|me p|us.
Summary
lnc|us|on oí the second ge| í||trot|on step eííect|ve|g removed |mpur|t|es ond contom|nonts to produce o pure torget prote|n.
2000
1500
0
1000
500
0 10 20 30 40 ml
A
280
mAU
100
120
50
A
280
mAU
0
0 20 40 60 80 100 120 ml
AC step:
Column: l3lTrop ¬l l m|
Sample: 4 m| l3l¯2-poromgos|n-δ-$o| |n c|or|ned E. coli |gsote
Flow rate: l m|/m|n
Binding buffer: 20 ml Tr|s-¬C|, 200 ml NoC|, l ml L0T^,
l ml 0TT, p¬ 7.4
Elution buffer: l0 ml mo|tose |n b|nd|ng buííer
GF step:
Column: ¬|Lood l6/60 $uperdex 200 pg
Sample: L|uted poo| |2 m|l írom l3lTrop ¬l l m|
Flow rate: l m|/m|n
Buffer: l3$ buííer, p¬ 7.4
97
66
45
30
20
14
(M
r
× 10
3
)
MBP*2-
paramyosin-
-Sal
L
M
W

m
a
r
k
e
r
s
S
a
m
p
l
e
,

d
i
l
u
t
e
d

1
:
1
5
F
l
o
w
t
h
r
o
u
g
h
,

d
i
l
u
t
e
d

1
:
1
5
,

M
B
P
T
r
a
p

H
P
M
B
P
T
r
a
p

H
P
H
i
L
o
a
d

S
u
p
e
r
d
e
x

1
6
/
6
0

2
0
0

p
g
Eluted pool
SDS-PAGE
40
M
B
P
-
t
a
g
g
e
d

p
r
o
t
e
i
n
s
Línc|ent two-step pur|ncot|on oí o
prote|n |nvo|ved |n metobo||c d|seose
Products featured: MBPTrap HP, Superdex 200 prep grade, XK 16/20, ÄKTAprime
lC^0 |l
r
85 500l |s o homotetromer prote|n |nvo|ved |n metobo||c d|seose. ln th|s studg, lC^0 wos pur|ned íor stob|||tg ío|d|ng ond
k|net|c stud|es. The pur|tg oí the e|uted íroct|ons wos determ|ned bg $0$-l^CL ono|gs|s. $ome truncoted íorms oí the torget prote|n,
os we|| os prote|n oggregotes were detected írom the nrst step ond were eííect|ve|g removed |n the second step.
Summary
The torget prote|n wos h|gh|g concentroted ond |t e|uted |n o smo|| vo|ume írom the í|rst oíí|n|tg step. Th|s removes the need to
concentrote the prote|n pr|or to ge| í||trot|on thus sov|ng t|me, cost, ond prote|n somp|e.
1000
1500
2000
2500
500
A280
mAU
0
0 20 40 60 80 100 ml 120
200
300
400
100
0
0 20 40 60 80 ml
A280
mAU
170
130
95
72
56
43
34
26
11
17
(M
r
× 10
3
)
MBP-
MCAD
M
o
l
e
c
u
l
a
r

w
e
i
g
h
t

m
a
r
k
e
r
s
S
t
a
r
t

m
a
t
e
r
i
a
l
,

d
i
l
u
t
e
d

1
:
6
F
l
o
w
t
h
r
o
u
g
h

M
B
P
T
r
a
p

H
P
,

d
i
l
u
t
e
d

1
:
6
E
l
u
t
e
d

f
r
a
c
t
i
o
n
s

f
r
o
m

M
B
P
T
r
a
p

H
P
E
l
u
t
e
d

f
r
a
c
t
i
o
n
s

f
r
o
m

S
u
p
e
r
d
e
x

2
0
0

p
g
AC step
Column: l3lTrop ¬l 5 m|
Sample: l5 m| oí N-term|no| l3l-lC^0 |n E. coli |gsote
Binding buffer: 20 ml Tr|s-¬C|, 200 ml NoC|, l ml L0T^,
l ml 0TT, p¬ 7.4
Elution buffer: l0 ml mo|tose |n b|nd|ng buííer
Flow rate: 5.0 m|/m|n |0.5 m|/m|n dur|ng somp|e |ood|ngl
System: ^KT^pr|me
GF step
Column: $uperdex 200 pg |n ×K l6/20
Sample: 2 m| oí e|uted íroct|on írom l3lTrop ¬l 5 m|
Buffer: 20 ml ¬LlL$, 200 ml NoC|, p¬ 7.0
Flow rate: 0.4 m|/m|n
System: ^KT^pr|me
SDS-PAGE
Acknowledgements: L. l. lo|er, 0r. von ¬ounersches K|ndersp|to|, lun|ch, Cermong
4l
M
B
P
-
t
a
g
g
e
d

p
r
o
t
e
i
n
s
Unottended outomoted
two-step pur|ncot|on
Products featured: MBPTrap HP, HiLoad 16/60 Superdex 200 pg, ÄKTAxpress
l3l-togged opopt|n prote|n |l
r
~ 60 000l wos pur|ned |n o two-step procedure us|ng ^KT^xpress w|th oínn|tg chromotogrophg |^Cl
ond ge| n|trot|on |Cfl protoco|s. The prote|n wos |ntended íor crgsto|||zot|on screen|ng ond íunct|ono| stud|es. The e|uted peok írom
the l3lTrop co|umn wos outomot|co||g co||ected |n o |oop ond |njected onto the ge| n|trot|on co|umn.
Summary
The use oí on|g two steps |n on outomoted protoco| w|th ^KT^xpress |ncreoses occurocg, o||ows íor honds-oíí operot|on, ond
e||m|notes humon error.
AC column: l3lTrop ¬l 5 m|
Sample: l5 m| oí l3l-opopt|n |n E. coli |gsote, l
r
~60 000
Flow rate: 5 m|/m|n
Binding buffer: 20 ml Tr|s-¬C|, 200 ml NoC|, l ml L0T^,
l ml 0TT, p¬ 7.4
Elution buffer: l0 ml mo|tose |n b|nd|ng buííer
GF column: ¬|Lood l6/60 $uperdex 200 pg
Sample: Co||ected poo| írom l3lTrop ¬l
Flow rate: 0.3 m|/m|n
Buffer: l0 ml sod|um phosphote, l40 ml NoC|, 0.5 l L0T^, p¬ 7.2
System: ^KT^xpress
Elution buffer
%B
0
500
1000
1500
2000
A
280
mAU
0
20
40
60
80
100
60 80 100 120 140 160 180 ml
AC GF
SDS-PAGE
Acknowledgements: R. ¿|mmermon, Le|den Un|vers|tg, 2333CC Le|den, The Nether|onds
250
130
100
70
55
35
27
15
10
(M
r
× 10
3
)
M
o
l
e
c
u
l
a
r

w
e
i
g
h
t

m
a
r
k
e
r
Eluted fractions from gel filtration
42
M
B
P
-
t
a
g
g
e
d

p
r
o
t
e
i
n
s
lncreos|ng the pur|ncot|on sco|e
Products featured: MBPTrap HP columns, XK 26/20 column, Dextrin Sepharose High Performance,
ÄKTAexplorer, ExcelGel SDS Gradient 8–18, Multiphor II Electrophoresis System, LMW-SDS Marker Kit
To |nvest|gote the reproduc|b|||tg oí sco|e-up us|ng l3lTrop co|umns, l3l2¯-β-go|octos|dose |l
r
~l58 000l, o recomb|nont togged
mu|t|mer, wos pur|ned on l3lTrop ¬l l m| ond 5 m| co|umns, wh|ch ore prepocked w|th 0extr|n $ephorose ¬|gh leríormonce
med|um. further sco|e-up wos períormed on on ×K 26/20 co|umn, pocked w|th the some chromotogrophg med|um. $omp|e |ood
wos |ncreosed nve-ío|d íor eoch sco|e-up step.
Summary
The co|umns gove comporob|e resu|ts w|th h|gh pur|tg ond s|m||or g|e|ds |obout 60%, doto not shownl, coní|rm|ng the eose
ond reproduc|b|||tg oí sco||ng up pur|í|cot|ons írom l3lTrop ¬l co|umns to on ×K 26/20 co|umn pocked w|th the some
chromotogrophg med|um.
Columns: l3lTrop ¬l l m|, l3lTrop ¬l 5 m|, 0extr|n $ephorose ¬|gh
leríormonce pocked |n ×K 26/20, 29 m|, bed he|ght 5.5 cm
Sample: l3l2¯-β-go|octos|dose |l
r
~l58 000l |n E. coli |gsote
Sample volumes: 5 m| |l3lTrop ¬l l m|l, 25 m| |l3lTrop ¬l 5 m|l,
l25 m| |×K 26/20 co|umnl
Binding buffer: 20 ml Tr|s-¬C|, 200 ml NoC|, l ml L0T^, l ml 0TT, p¬ 7.4
Elution buffer: l0 ml mo|tose |n b|nd|ng buííer
Flow rates: l3lTrop ¬l l m|. l.0 m|/m|n |0.5 m|/m|n dur|ng somp|e |ood|ngl
l3lTrop ¬l 5 m|. 5.0 m|/m|n |2.5 m|/m|n dur|ng somp|e |ood|ngl
×K 26/20 co|umn. l3 m|/m|n
System: ^KT^exp|orer
0
500
1000
1500
2000
2500
3000
A
280
mAU
0.0 5.0 10.0 15.0 20.0 ml
80
100
Elution Buffer %
60
40
20
0
0
500
1000
1500
2000
2500
3000
0 100 200 300 400 500 ml
80
100
60
40
20
0
600
A
280
mAU
Elution Buffer %
97
66
45
30
20
14
(M
r
× 10
3
)
MBP2*--
galactosidase
L
M
W

m
a
r
k
e
r
s
M
B
P
T
r
a
p

H
P

1

m
l
,

d
i
l
u
t
e
d

1
:
6
M
B
P
T
r
a
p

H
P

5

m
l
,

d
i
l
u
t
e
d

1
:
1
2
X
K

2
6
/
2
0
,

d
i
l
u
t
e
d

1
:
1
2
M
B
P
T
r
a
p

H
P

1

m
l
,

d
i
l
u
t
e
d

1
:
3
M
B
P
T
r
a
p

H
P

5

m
l
,

d
i
l
u
t
e
d

1
:
3
X
K

2
6
/
2
0
,

d
i
l
u
t
e
d

1
:
3
S
t
a
r
t

m
a
t
e
r
i
a
l
,

d
i
l
u
t
e
d

1
:
3
Flowthrough Eluted pool
MBPTrap HP 1 ml Dextrin Sepharose High Performance XK 26/20
SDS-PAGE
43
M
B
P
-
t
a
g
g
e
d

p
r
o
t
e
i
n
s
0rder|ng |níormot|on
Purification
l3lTrop ¬l 5 × l m| 28-9l87-78
l × 5 m| 28-9l87-79
5 × 5 m| 28-9l87-80
0extr|n $ephorose ¬|gh
leríormonce
25 m| 28-9355-97
l00 m| 28-9355-98
Purification
¬|Lood l6/60 $uperdex 30 pg l × l20 m| l7-ll39-0l
¬|Lood 26/60 $uperdex 30 pg l × 320 m| l7-ll40-0l
¬|Lood l6/60 $uperdex 75 pg l × l20 m| l7-l068-0l
¬|Lood 26/60 $uperdex 75 pg l × 320 m| l7-l070-0l
¬|Lood l6/60 $uperdex 200 pg l × l20 m| l7-l069-0l
¬|Lood 26/60 $uperdex 200 pg l × 320 m| l7-l07l-0l
Lxce|Ce| $0$ Crod|ent 8-l8 6 80-l255-53
Llv-$0$ lorker K|t l0 v|o|s l7-0446-0l
$uperdex 200 prep grode l50 m| l7-l043-0l
×K l6/20 emptg co|umn l l8-8773-0l
Product Quantity Code No.
For more information on Biacore systems and chromatography and/or electrophoresis systems, please visit www.gelifesciences.com
44
0uo|-togged prote|ns
Recomb|nont prote|ns con be des|gned to conto|n N- or C-term|no| oínn|tg
togs. The |nc|us|on oí two d|ííerent togs |n o construct resu|ts |n o torget
prote|n thot con be pur|ned w|th d|ííerent oínn|tg chromotogrophg med|o
thus produc|ng o purer prote|n w|th o stro|ghtíorword pur|ncot|on scheme.
lí d|ííerent togs ore pos|t|oned on eoch end oí the prote|n, o duo|-togged
pur|ncot|on scheme wou|d normo||g produce o íu||-|ength prote|n. The
oínn|tg tog con be c|eoved |í c|eovoge s|tes ore |nc|uded between the tog
ond the torget prote|n.
45
D
u
a
l
-
t
a
g
g
e
d

p
r
o
t
e
i
n
s
0ne versus two oínn|tg steps
Products featured: HisTrap HP, GSTrap 4B, ÄKTAxpress, ExcelGel SDS Gradient 8–18, Multiphor II
Electrophoresis System, LMW-SDS Marker Kit
To compore pur|ncot|on us|ng e|ther one tog or o comb|not|on oí two d|ííerent togs, o duo|-togged green fuorescent prote|n
|¦¬|s¦
6
-Cfl-C$Tl wos pur|ned |n three d|ííerent wogs. lur|ncot|on on ¬|sTrop ¬l showed thot the med|um hod oínn|tg íor other
not|ve prote|ns w|th exposed h|st|d|ne res|dues os we||, whereos ut|||z|ng the h|gh spec|nc|tg oí the C$T tog íor the g|utoth|one
||gond on C$Trop 43 gove o much purer prote|n. The comb|not|on oí the two togs produced the h|ghest pur|tg.
Summary
0uo|-togged pur|í|cot|on coníers o sgnerg|st|c beneí|t |h|gher pur|tgl thot somet|mes e|udes s|ng|e-togged pur|í|cot|on schemes.
Sample: |¬|sl
6
-Cfl-C$T |n E.coli |gsote
Columns:
Affinity chromatography: ¬|sTrop ¬l 5 m| ond C$Trop 43 5 m|
Desalting: ¬|lrep 26/l0 0eso|t|ng
Buffers:
HisTrap HP
Binding buffer: 20 ml l3$, 20 ml |m|dozo|, 0.5 l NoC|, p¬ 7.4
Elution buffer: 20 ml l3$, 500 ml |m|dozo|, 0.5 l NoC|, p¬ 7.4
GSTrap 4B:
Binding buffer: l0 ml l3$, p¬ 7.4
Elution buffer: 50 ml Tr|s, 20 ml reduced g|utothone, p¬ 8
HiPrep 26/10 Desalting:
Buffer: 50 ml Tr|s-¬C| , l50 ml NoC|, p¬ 7.5
HiPrep™ 16/10
HiPrep™ 16/10
HiPrep™ 16/10
HiPrep™ 16/10
Run 1 Run 2 Run 3
HisTrap HP HiPrep 26/10
Desalting
GSTrap 4B HiPrep 26/10
Desalting
GSTrap 4B HiPrep 26/10
Desalting
HisTrap HP HiPrep 26/10
Desalting
96
66
45
30
20
14
(M
r
× 10
3
)
L
M
W

m
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2
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3
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3
(His)
6
-GFP-GST
SDS-PAGE
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p
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46
lncreosed pur|tg w|th
duo|-togged expressed prote|n
Products featured: HisTrap HP, StrepTrap HP, ÄKTAxpress
$|nce o h|gh degree oí pur|tg |s cruc|o| íor successíu| íunct|ono| stud|es, pur|tg resu|ts oí the two-step method were compored w|th
those írom s|ng|e-step pur|ncot|ons. ^ duo|-togged Strep tog ll-|h|st|d|nel
6
prote|n |l
r
~l5 400l expressed |n E. coli wos pur|ned íor
method deve|opment us|ng o two-step procedure compr|s|ng |mmob|||zed meto| oínn|tg chromotogrophg |ll^Cl ío||owed bg oínn|tg
chromotogrophg on o $trepTrop ¬l co|umn. ^|| the exper|ments were conducted ot 4ºC to preserve prote|n stob|||tg.
Summary
$0$-l^CL resu|ts c|eor|g demonstrote the beneí|ts oí o duo|-togged opprooch to prote|n pur|í|cot|on, espec|o||g when h|gh
pur|tg |s requ|red.
188
98
62
49
38
28
(M
r
× 10
3
)
17
14
6
3
Dual-tagged
Strep-tag II-(His)
6
M
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m
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o
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p
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p
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p
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F
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h
Individual
HisTrap HP
HisTrap HP +
StrepTrap HP
Individual
StrepTrap HP
Sample: l5 m| Strep-tog ll-|h|st|d|nel
6
prote|n
|l
r
~l5 400l |n E. coli |gsote
Individual HisTrap HP purification
Column: ¬|sTrop ¬l l m|
Binding buffer: 20 ml sod|um phosphote,
500 ml NoC|, 20 ml |m|dozo|e, p¬ 7.5
Elution buffer: 20 ml sod|um phosphote,
500 ml NoC|, 500 ml |m|dozo|e, p¬ 7.5
Flow rate: 0.8 m|/m|n
Individual StrepTrap HP purification
Column: $trepTrop ¬l l m|
Binding buffer: l00 ml Tr|s-¬C|, l50 ml NoC|,
l ml L0T^, p¬ 8.0
Elution buffer: 2.5 ml desth|ob|ot|n |n
b|nd|ng buííer
Flow rate: 0.8 m|/m|n
Two-step HisTrap HP and StrepTrap HP purification
Column: ¬|sTrop ¬l l m|
Binding buffer: 20 ml sod|um phosphote,
500 ml NoC|, 20 ml |m|dozo|e, p¬ 7.5
Elution buffer: 20 ml sod|um phosphote,
500 ml NoC|, 500 ml |m|dozo|e, p¬ 7.5
Flow rate: 0.8 m|/m|n
Column: $trepTrop ¬l l m|
Sample: L|uted íroct|on írom ¬|sTrop ¬l, l m|
Binding buffer: l00 ml Tr|s-¬C|, l50 ml NoC|, l ml
L0T^, p¬ 8.0
Elution buffer: 2.5 ml desth|ob|ot|n |n b|nd|ng buííer
Flow rate: 0.2 m|/m|n
System: ^KT^xpress
SDS-PAGE
Acknowledgements: l. N||sson, R. $vensson ond L. ¬o|mgren, 3|ov|trum, $tockho|m, $weden
47
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p
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n
s
lmpoct oí revers|ng the order
oí oínn|tg pur|ncot|on
Products featured: StrepTrap HP, HisTrap HP, ÄKTAxpress, ExcelGel SDS Gradient 8–18, Multiphor II
Electrophoresis System, LMW-SDS Marker Kit
To evo|uote whether the order oí oínn|tg co|umns |n o chosen pur|ncot|on method motters w|th respect to g|e|d ond pur|tg. |¬|sl
6
-
mCherrg-Strep-tog ll duo|-togged torget prote|n, wos pur|ned us|ng $trepTrop ¬l ío||owed bg ¬|sTrop ¬l ond subsequent|g |n the
reverse order. $0$-l^CL ono|gs|s showed thot the pur|tg oí the prote|n wos the some, |rrespect|ve oí the order oí oínn|tg pur|ncot|on
used. ^n N-term|no||g truncoted torget prote|n |dent|ned bg moss spectrometrg |doto not shownl, however, possed through the
¬|sTrop ¬l co|umn more s|ow|g thon nontogged prote|ns, wh|ch occounts íor the two peoks observed |n the fowthrough.
Columns: $trepTrop ¬l l m|, ¬|sTrop ¬l l m|
Sample: |¬|sl
6
-mCherrg-Strep-tog ll |n E. coli |gsote
Binding buffer (StrepTrap HP): l00 ml Tr|s-¬C|, l50 ml NoC|,
l ml L0T^, p¬ 8.0
Elution buffer (StrepTrap HP): l00 ml Tr|s-¬C|, l50 ml NoC|,
l ml L0T^, 2.5 ml desth|ob|ot|n, p¬ 8.0
Binding buffer (HisTrap HP): 20 ml phosphote, 500 ml NoC|,
5 ml lm|dozo|e, p¬ 7.4
Elution buffer (HisTrap HP): 20 ml phosphote, 500 ml NoC|,
500 ml lm|dozo|e, p¬ 7.4
System: ^KT^xpress
Summary
The some omount |0.9 mgl oí prote|n wos produced |n both exper|ments ond the pur|tg oí the torget prote|n wos not oííected
bg the order oí the oíí|n|tg co|umns used.
0
100
200
300
400
500
600
700
mAU
70 80 90 100 110 ml
A
280
StrepTrap HP
elution
HisTrap HP
elution
HisTrap HP
flow through
66
30
M
r
× 10
3
96
45
20.1
14.4
(His)
6
-mCherry-Strep-tag II
0
500
1000
1500
2000
mAU
70 80 90 100 110 120 130 140 ml
A
280
HisTrap HP
elution
StrepTrap HP
elution
Loop
wash
StrepTrap HP
flow through
M
r
× 10
3
96
45
20.1
14.4
66
30
(His)
6
-mCherry-Strep-tag II
for contoct |níormot|on íor gour |oco| oínce,
p|eose v|s|t. www.ge||íesc|ences.com/contoct
CL ¬eo|thcore 3|o-$c|ences ^3
3jorkgoton 30
75l 84 Uppso|o
$weden
www.ge||íesc|ences.com
imagination at work
CL, |mog|not|on ot work ond CL monogrom ore trodemorks oí
Cenero| L|ectr|c Compong.
^KT^des|gn, ^KT^exp|orer, ^KT^pr|me, ^KT^xpress, ^mershom,
3|ocore, LCL, Lxce|Ce|, Crov|Trop, C$Tprep, C$Trop, ¬|Lood,
¬|sTrop, ¬|slrep, ¬|lrep, ¬gbond, ¬gpern|m, l3lTrop,
lu|t|Trop, lhostCe|, lre$c|ss|on, RL$0URCL, $ephorose,
$uperdex, $p|nTrop, $trepTrop, ond UNlC0RN ore trodemorks
oí CL ¬eo|thcore compon|es.
¬|st|d|ne-togged prote|ns. lur|ncot|on ond preporot|on oí
íus|on prote|ns ond oínn|tg pept|des compr|s|ng ot |eost two
odjocent h|st|d|ne res|dues mog requ|re o ||cense under U$
potent numbers 5,284,933 ond 5,3l0,663, ond equ|vo|ent
potents ond potent opp||cot|ons |n other countr|es |oss|gnee.
¬oíímon Lo Roche, lncl.
N| $ephorose 6 fost f|ow. Th|s products |s covered bg U$ pot
No 6 623 655 ond the|r equ|vo|ents |n other countr|es.
pCL× vectors ore to be used íor sc|ent|nc |nvest|got|on
ond reseorch ond íor no other purpose whotsoever ond o
||cense íor commerc|o| use oí the ||censed products ond the
processes c|o|med |n U$ potent 5,654,l76 ond equ|vo|ent
potents ond potent opp||cot|ons |n other countr|es must be
negot|oted d|rect|g w|th l||||pore Corp |íormer|g Chem|con
lnternot|ono| lncl bg the purchoser pr|or to such use.
$trepTrop ¬l ond $trepToct|n $ephorose ¬|gh leríormonce
These products ore covered bg U$ potent number 6,l03,493
ond equ|vo|ent potents ond potent opp||cot|ons |n other
countr|es. The purchose oí $trepTrop ¬l ond $trepToct|n
$ephorose ¬|gh leríormonce |nc|udes o ||cense under such
potents íor non-pront ond |n-house reseorch on|g. l|eose
contoct l3^ ||nío©|bo-go.coml íor íurther |níormot|on on
||censes íor commerc|o| use oí $trepToct|n.
^|| th|rd portg trodemorks ore the propertg oí the|r respect|ve
owners.
© 2008 Cenero| L|ectr|c Compong-^|| r|ghts reserved.
f|rst pub||shed ^ugust 2008
^|| goods ond serv|ces ore so|d subject to the terms ond
cond|t|ons oí so|e oí the compong w|th|n CL ¬eo|thcore
wh|ch supp||es them. ^ copg oí these terms ond cond|t|ons
|s ovo||ob|e on request. Contoct gour |oco| CL ¬eo|thcore
representot|ve íor the most current |níormot|on.
CL ¬eo|thcore 3|o-$c|ences ^3
3jorkgoton 30
75l 84 Uppso|o
$weden
CL ¬eo|thcore Lurope, Cmb¬
lunz|nger $trosse 5
0-79lll fre|burg
Cermong
CL ¬eo|thcore 3|o-$c|ences Corp.
800 Centenn|o| ^venue, l.0. 3ox l327
l|scotowog, NJ 08855-l327
U$^
CL ¬eo|thcore 3|o-$c|ences KK
$onken 3|dg., 3-25-l, ¬gokun|ncho
$h|njuku-ku, Tokgo l69-0073
Jopon
28-9353-64 ^^ 08/2008


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U
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2
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Strep Strep

Cloning

Cell culture

Screening

Scale-up purification

Structural & functional studies

Lead design

E. coli.

GST-tagged proteins pGEX-1 T (27-4805-01) Thrombin Protease Schistosoma japonicum E. coli Leu Val Pro Arg Gly Ser Pro Glu Phe Ile Val Thr Asp CTG GTT CCG CGT GGA TCC CCG GAA TTC ATC GTG ACT GAC TGA CGA Stop codons EcoR I BamH I pGEX-2T (27-4801-01) Thrombin Protease Leu Val Pro Arg Gly Ser Pro Gly Ile His Arg Asp CTG GTT CCG CGT GGA TCC CCG GGA ATT CAT CGT GAC TGA CTG ACG Stop codons BamH I Sma I EcoR I pGEX-2TK (27-4587-01) Thrombin Protease Kinase Leu Val Pro Arg Gly Ser Arg Arg Ala Ser Val CTG GTT CCG CGT GGA TCT CGT CGT GCA TCT GTT GGA TCC CCG GGA ATT CAT CGT GAC TGA Stop codons BamH I Sma I EcoR I pGEX-4T-1 (27-4580-01) Thrombin Protease Leu Val Pro Arg Gly Ser Pro Glu Phe Pro Gly Arg Leu Glu Arg Pro His Arg Asp CTG GTT CCG CGT GGA TCC CCG GAA TTC CCG GGT CGA CTC GAG CGG CCG CAT CGT GAC TGA Stop codons Not I EcoR I Sma I Sal I Xho I BamH I pGEX-4T-2 (27-4581-01) Thrombin Protease Leu Val Pro Arg Gly Ser Pro Gly Ile Pro Gly Ser Thr Arg Ala Ala Ala Ser CTG GTT CCG CGT GGA TCC CCA GGA ATT CCC GGG TCG ACT CGA GCG GCC GCA TCG TGA Stop codon BamH I Not I EcoR I Sma I Sal I Xho I (27-4583-01) Thrombin Protease Leu Val Pro Arg Gly Ser Pro Asn Ser Arg Val Asp Ser Ser Gly Arg Ile Val Thr Asp CTG GTT CCG CGT GGA TCC CCG AAT TCC CGG GTC GAC TCG AGC GGC CGC ATC GTG ACT GAC TGA Stop codons BamH I Not I EcoR I Sma I Sal I Xho I pGEX-3X (27-4803-01) Factor Xa Protease Ile Glu Gly Arg Gly Ile Pro Gly Asn Ser Ser ATC GAA GGT CGT GGG ATC CCC GGG AAT TCA TCG TGA CTG ACT GAC Stop codons BamH I Sma I EcoR I pGEX-5X-1 (27-4584-01) Factor Xa Protease Ile Glu Gly Arg Gly Ile Pro Glu Phe Pro Gly Arg Leu Glu Arg Pro His Arg Asp ATC GAA GGT CGT GGG ATC CCC GAA TTC CCG GGT CGA CTC GAG CGG CCG CAT CGT GAC TGA Stop codons BamH I EcoR I Sma I Sal I Xho I Not I pGEX-4T-3 pGEX-5X-2 (27-4585-01) Factor Xa Protease Ile Glu Gly Arg Gly Ile Pro Gly Ile Pro Gly Ser Thr Arg Ala Ala Ala Ser ATC GAA GGT CGT GGG ATC CCC GGA ATT CCC GGG TCG ACT CGA GCG GCC GCA TCG TGA Stop codon BamH I Not I EcoR I Sma I Sal I Xho I pGEX-5X-3 (27-4586-01) Factor Xa Protease Ile Glu Gly Arg Gly Ile Pro Arg Asn Ser Arg Val Asp Ser Ser Gly Arg Ile Val Thr Asp ATC GAA GGT CGT GGG ATC CCC AGG AAT TCC CGG GTC GAC TCG AGC GGC CGC ATC GTG ACT GAC TGA Stop codons EcoR I Sma I Sal I Xho I BamH I Not I pGEX-6P-1 (27-4597-01) PreScission Protease Leu Glu Val Leu Phe Gln Gly Pro Leu Gly Ser Pro Glu Phe Pro Gly Arg Leu Glu Arg Pro His CTG GAA GTT CTG TTC CAG GGG CCC CTG GGA TCC CCG GAA TTC CCG GGT CGA CTC GAG CGG CCG CAT BamH I EcoR I Sma I Sal I Xho I Not I pGEX-6P-2 (27-4598-01) PreScission Protease Leu Glu Val Leu Phe Gln Gly Pro Leu Gly Ser Pro Gly Ile Pro Gly Ser Thr Arg Ala Ala Ala Ser CTG GAA GTT CTG TTC CAG GGG CCC CTG GGA TCC CCA GGA ATT CCC GGG TCG ACT CGA GCG GCC GCA TCG BamH I EcoR I Sma I Sal I Xho I Not I pGEX-6P-3 (27-4599-01) PreScission Protease Leu Glu Val Leu Phe Gln Gly Pro Leu Gly Ser Pro Asn Ser Arg Val Asp Ser Ser Gly Arg CTG GAA GTT CTG TTC CAG GGG CCC CTG GGA TCC CCG AAT TCC CGG GTC GAC TCG AGC GGC CGC BamH I EcoR I Sma I Sal I Xho I Not I Bal I at ut ferase rans S-t ne hio Tth111 I Aat II Ptac BspM I gl p Am pSj10 Bam7Stop7 r Pst I pGEX ~4900 bp Nar I EcoR V BssH II Apa I BstE II Mlu I pBR322 ori la c q I p4.5 AlwN I .

GST-tagged proteins Glutathione Sepharose * Purification modules also contain buffers .

coli Average yield of eluted protein (μg) 500 450 400 350 300 250 200 0 20 40 Yield GST-hippocalcin. ExcelGel SDS Gradient 8–18.GST-tagged proteins Products featured: GST MultiTrap FF and GST MultiTrap 4B. coli 96-well filter plates: Sample: E. GST MultiTrap 4B Sample preparation: Binding buffer: Elution buffer: Elution method: 60 80 SDS-PAGE 120 min 60 min (Mr × 103) 100 120 140 Incubation time (min) 30 min 10 min 3 min 0 min 97 66 45 30 20 14 GST-hippocalcin LM W Summary Sta l wt hro ug h Elu Flo at e wt hro ug h Elu Flo ate wt hro ug h Elu Flo ate wt hro ug h Elu ate Flo wt hro ug h Elu Flo ate wt hro ug h Elu at e rs rke ma rt m Flo ate ria . GST MultiTrap FF Yield GST-hippocalcin. Multiphor II Electrophoresis System. LMW-SDS Marker Kit E.

GST-tagged proteins Products featured: GST SpinTrap Purification Module. GST Detection Module 96-well plate .

4 0.GST-tagged proteins Column: Sample: β Elution buffer: 0.8 Solubilized material Flowthrough Eluate Activity (A340/min*ml) Binding buffer: 0.2 0 DD M OG CH AP S 20 en we T Tr it X on -1 00 o rc Sa sy l SDS-PAGE DDM (Mr × 103) OG CHAPS Tween 20 Triton X-100 Sarcosyl 94 67 43 GST-ecoKch Acknowledgements: Summary rke rs hro ug GS h TS pin Elu Tra ate Flo p wt fro hro m ug GS h TS pin Elu Tra ate Flo p wt fro hro m GS ug TS h pin Elu Tra Flo ate p wt fro hro m ug GS h TS pin Elu Tra ate Flo p wt fro hro m ug GS h TS pin Elu Tra Flo ate p wt fro hro m ug GS h TS pin Tra p Flo ate fro m wt LM Elu W ma .6 0.

5 2. ÄKTAexplorer. ExcelGel SDS Gradient 8–18.5 0 5.5 3.7 mg pure GSTtagged protein Elution buffer % Elution buffer 100 30 20 14 ate Elu ma rt m 40 20 0 Summary Sta LM 60 ted W fra cti 80 rke ria on rs l .0 10.0 1. Multiphor II Electrophoresis System.0 20.GST-tagged proteins Products featured: GSTrap FF.5 1. coli SDS-PAGE (Mr × 103) 97 66 45 A280 3.0 ml Wash 2.0 15.0 0.0 2. LMW-SDS Marker Kit Column: Sample: Binding buffer: Elution buffer: Flow: System: E.

ÄKTAxpress. ExcelGel SDS Gradient 8–18. coli SDS-PAGE (Mr × 103) 97 66 45 30 A280 mAU 250 200 150 100 GST-hippocalcin Dimers of GST-hippocalcin 20 14 Large aggregates of GST-hippocalcin AC ) h( t ed fra cti on so fd rs ria l ma rke rt m ate 1500 1000 500 0 50 AC 100 150 GF 200 ml Summary Elu Flo wt 110 120 130 140 150 ml Sta LM A280 mAU W 0 hro ug 50 im er (GF ) . HiLoad 16/60 Superdex 200 pg. LMW-SDS Marker Kit Columns: Sample: Sample volume: Binding buffer (AC): Elution buffer (AC): Buffer (GF): Flow rates: System: E.GST-tagged proteins Products featured: GSTrap 4B. Multiphor II Electrophoresis System.

9 mg GSTPrep FF 16/10 Elution buffer %B 100 A 280 mAU 1400 1200 Elution buffer mAU 1200 1000 800 600 400 200 0 0 Elution buffer %B 100 111.GST-tagged proteins Products featured: pGEX-2T. GSTPrep FF 16/10. ÄKTAexplorer.0 mg 80 60 40 1000 800 600 400 Wash 80 60 40 20 800 600 400 60 40 20 0 20 200 0 200 0 0 10 20 30 ml 0 0 50 100 150 ml 0 100 200 300 400 500 600 ml Columns: Sample: Sample volumes: Binding buffer: Elution buffer: Flow rates: E. GSTrap FF. LMW-SDS Marker Kit GSTrap FF 1 ml A 280 Elution buffer Wash GSTrap FF 5 ml Elution buffer %B 100 5. Multiphor II Electrophoresis System. ExcelGel SDS Gradient 8–18. coli SDS-PAGE GSTrap FF 1 ml GSTrap FF 5 ml GSTPrep FF 16/10 (Mr × 10 ) 3 97 66 45 30 20 GST-DemA System: 14 Summary Elu ria l hro ug ted h fra cti Sta on s rt m ate ria Flo l wt hro Elu ug ted h fra cti Sta on rt m s ate Flo ria wt l hro Elu ug ted h fra cti on s Flo wt rke rs ma LM W Sta rt m ate .5 mg 80 A 280 mAU 1400 1200 1000 Wash Elution buffer 20.

GST-tagged proteins Sepharose Glutathione GST Cleavage site Recombinant protein Cleavage enzymes Factor Xa protease: Thrombin protease: PreScission Protease: .

GST-tagged proteins Products featured: GSTrap FF. ÄKTAxpress 100 90 80 Cleaved protein (%) 70 60 50 40 30 20 10 0 0 2 4 6 8 10 12 14 Incubation time (h) 10 units PreScission/mg protein 20 units PreScission/mg protein 40 units PreScission/mg protein Summary . PreScission Protease.

0 15.0 4.GST-tagged proteins Products featured: GSTrap FF.0 2. Multiphor II Electrophoresis System. se Tt rke va rs .0 6.0 0. tei Cle n. LMW-SDS Marker Kit A 280 3.0 10. Thrombin protease. ExcelGel SDS Gradient 8–18.5 3. pro pro tei dp ge d ag ge arg et ag T-t T-t Summary GS T -fr ee t GS GS Th rom bin pro tea n. Wash Target protein % Elution buffer 100 80 Free GST 60 40 20 0 Column: Sample: Binding buffer: Elution buffer: Flow rate: System: E.5 1.0 1.5 0 5.0 8.0 min Wash Incubation 16 h room temp.0 min 2. GSTrap FF 1 ml (Mr × 103) 97 66 45 30 20 14 Target protein ge rs ria l n n ) ml U/ (20 LM W ma rke lum lum ag ate cle a l co l co ma rt m 1m 5m ed W GS Sta LM 16 h av rot ein . ÄKTAexplorer.0 12. coli SDS-PAGE Thrombin protease cleavage.5 2.0 10.

GST-tagged proteins Products featured: pGEX-6P. ExcelGel SDS gradient 8–18. HiLoad 16/60 Superdex 75 pg. ÄKTAxpress. LMW-SDS Marker Kit Columns: Sample: α A 280 mAU Cleaved protein Binding/cleavage buffer (AC): Elution buffer (AC): Buffer (GF): System: 2000 Regeneration 1500 46 mg 1000 500 SDS-PAGE 0 (Mr × 103) 200 AC 250 GF 300 ml 97 66 45 pur 30 20 14 ma rke rs Sta rt m ate ria l Flo Pu wt rifi hro ed ug . cl h ea ve dG ST -pu Un r cle av ed GS T-p ur Summary LM W . PreScission Protease. Multiphor II Electrophoresis System. GSTrap HP.

GST 96-well Detection Module E.GST-tagged proteins Products featured: pGEX-6P-1. coli Control 1 2 3 4 5 Anti-luciferase detection 6 7 8 9 10 11 12 Columns Summary .

Amersham Hyperfilm ECL. co li + SDS-PAGE nic pG EX -5X E.GST-tagged proteins E. co So Products featured: Anti-GST Antibody.E ate . Amersham Hybond ECL. coli nic ate .E So E. co . Amersham GST Western Blotting Detection Kit Summary E. coli Pre sta ine dM W ma rke 14 20 30 45 97 66 HRP .E ate So nic -Lu ce xp re ssi ng GS res s Pu rifi ed GS T r (Mr × 103) So nic . coli So nic ate . li + . co pG EX -4T -E7 exp ate . KL 45 cel l s (D na K ov ere xp res s ing ) -1 e EX -5X ras 7 ife T-E pG uc GS li + T-l ing li T G1 .

Protein expression Detection λ Tag cleavage All vectors include BL21 Related products Purification † available by specific customer order For more information on equipment for chromatography and/or electrophoresis.com .GST-tagged proteins Product Quantity Code No.gelifesciences. Product Quantity Code No. please visit www.

.

Histidine-tagged proteins Ni Sepharose .

row 2 1 2 3 4 5 6 1 2 3 4 5 6 M Elution. row 4 Elution. row 5 1 2 3 4 5 6 1 2 3 4 5 6 M Elution. row 3 1 2 3 4 5 6 M Elution. row 1 Elution. row 6 Elution.Histidine-tagged proteins Products featured: His MultiTrap HP 96-well filter plate: Samples: E. coli 100 75 50 37 Equilibration buffer: Wash buffer: Elution buffer: Liquid handling station: Vacuum station: 25 20 15 10 P : Protein purification (37 kDa) B : Blank Clarified bacterial extracts (Mr × 10 ) 100 75 50 37 25 20 15 10 3 1 2 3 4 5 6 M Elution. row 7 1 2 3 4 5 6 1 234 5 6 Acknowledgements: Summary . coli (Mr × 103) 150 M B P B P B P B P B P B P B P B P B P B P B P B P B E.

Histidine-tagged proteins Products featured: His SpinTrap. LMW-SDS Marker Kit Column: Sample: E. dil ute d1 :2 ida 50 mM zol ed 5m M im Sta rt m ate LM W . uri dil ng ute bin d1 din :2 g. d uri ilu im ng ted ida bin 10 zol 1:1 0m din ed 0 g. ExcelGel SDS Gradient 8–18. M uri dil im ng ute ida bin zol d1 din ed :2 g. M uri dil im ng ute ida bin 20 zol d1 0m din ed :2 g. Multiphor II Electrophoresis System.coli SDS-PAGE Eluted pool Binding/wash buffer: (Mr × 103) Elution buffer: 97 66 45 30 20 14 APB 7-(His)6 Summary ma rke ria rs l.

Amersham Hybond ECL. d ilu Flo ted wt hro 1:2 0 ug h. dil ute d1 :20 1 2 ) 09 ate ate ate te dJ M1 JM 1 me d for Ne ga ti ve co n tro l (un tra ns 09 ) Elu a Elu Summary Ne ga tiv ec on tro l (u ntr an s for me Elu Elu . Amersham High-Range Rainbow Molecular Weight Markers E. dil ut e d1 :20 1 2 Hig hm ole cu lar we igh Sta tm rt m ark ate ers ria l.Histidine-tagged proteins Products featured: His GraviTrap Kit. coli Column: Sample: Binding/wash buffer: Elution buffer: E. ECL Mouse IgG.coli SDS-PAGE (Mr × 103) Western blot 220 97 66 45 30 20 14 Hig hm ole cu lar we igh Sta tm rt m ark ate ers ria l. d ilu Flo ted wt hro 1:2 0 ug h. Anti-His Antibody. PhastSystem. HRP-linked Whole Ab. PhastGel Gradient 10–15.

mark e l. coli A280 3. 1 dilu 0 m ted rs M 1:1 im ida 0 zol Wa e sh 1.0 2.5 2.0 1.5 1.5 0 0 Wash 1 Wash 2 Wash 3 Elution 1 Elution 2 Imidazole concentration (mM) Elution 3 500 400 300 200 100 5 10 15 20 0 25 30 Elution volume (ml) SDS-PAGE (Mr × 103) 97 66 45 30 20 14 MBP-(His)6 e rifi Wa ed sa LMW m sh 5 m ple. 3.Histidine-tagged proteins Products featured: HisTrap FF crude Kit. LMW-SDS Marker Kit Column: Sample: Flow rate: Buffers: Fraction size: Equipment: E. ExcelGel SDS Gradient 8–18. Multiphor II Electrophoresis System. 50 0m M im i im im im da zol e .0 0. 10 mM im ida zol e e e e da zol zol ida zol ida zol im i ida mM mM M M 0m 0m 30 50 10 30 2. sh Wa sh Elu tio n Elu tio n Un cla Summary Elu tio n Wa 3. 2. 1.

ÄKTAprime plus Sample: Column: Binding buffer: Elution buffer: System: E.5 80 2.5 0 5 10 15 20 25 30 35 40 20 0 min Summary .Histidine-tagged proteins Products featured: HisTrap HP.0 0.5 1.0 60 1. coli A280 Elution Buffer % 100 2.

ial ma rk ate r Sta rt m po ol. dil u LM W Summary Elu ted dil ute d1 :4 . ExcelGel SDS Gradient 8–18.Histidine-tagged proteins Pichia pastoris Products featured: HisTrap FF crude. pastoris 3000 2000 1000 0 0 100 200 300 400 ml 50 Flow rate: System: 0 SDS-PAGE (Mr × 103) 97 66 45 30 20 14 (His)6-YNR064c ers 1:2 t ed Elu t ed po ol. ÄKTAexplorer. Multiphor II Electrophoresis System. LMW-SDS Marker Kit Pichia pastoris A280 mAU 5000 4000 Elution Buffer % 100 Column: Sample: myces cerevisiae Binding buffer: Elution buffer: SaccharoP.

5 92 92.5 94 94.Histidine-tagged proteins Products featured: HisTrap HP. ÄKTAexplorer. coli. coli Sample: Column: Solubilizing buffer: Denatured binding buffer: Refolding buffer: Native binding buffer: Native elution buffer: Flow rate: System: Pooled fraction from the purification of refolded scFv 57P antibody fragment A280 mAU 500 400 300 200 100 0 91.5 93 93. E.5 95 ml 80 60 40 20 0 Elution buffer % 100 SPR sensogram 20 000 Response signal (RU) 15 000 10 000 5000 0 -5000 200 400 600 800 1000 1200 1400 1600 s pool Reference cell Sample cell Response difference after subtraction of reference cell A280 mAU 500 3 5 Elution buffer % 100 80 400 300 1 200 4 100 0 0 20 40 60 80 100 ml 2 20 60 40 0 Summary . Biacore System E.

un cle av ed rke ug 04 AP C1 0 20 14 IEX elution buffer: ma rt m W Sta Flo AP C1 04 wt 0a fte hro GF buffer: LM ate l A280 mAU Cleaved protein Elution buffer % 100 80 cle av ed Pu rifi e d. 1500 1000 Regeneration 60 40 500 11 mg 20 0 1100 AC B10 0 ml 1200 DS 1300 1400 IEX 1500 GF Summary rA C– . coli (Mr × 103) 97 66 45 Uncleaved APC1040 (Mr 38 900) Cleaved APC1040 (Mr 36 400) 30 AC binding buffer: AC cleavage buffer: AC elution buffer: DS and IEX binding buffer: rs h ria GF X– –IE DS Co ntr ol. HiLoad 16/60 Superdex 75 pg. LMW-SDS Marker Kit Sample: Columns: AC: DS: IEX: GF: Cleavage conditions: E. Multiphor II Electrophoresis System.Histidine-tagged proteins Products featured: HisTrap HP. HiPrep 26/10 Desalting. ExcelGel SDS Gradient 8–18. ÄKTAxpress. RESOURCE Q.

ExcelGel SDS Gradient 8–18.0 30. ÄKTAexplorer. Multiphor II Electrophoresis System. HisPrep FF 16/10.2 mg 50 0 15. LMW-SDS Marker Kit HisTrap FF 1 ml A280 mAU 4000 3500 3000 2500 2000 1500 1000 500 0 0.0 ml 0 150 ml 0 500 600 700 ml Columns: Sample: Sample volumes: Binding buffer: Elution buffer: E.0 20.0 5 .0 25.0 50 100 Elution buffer % 100 33 mg 50 HisPrep FF 16/10 A280 mAU 4000 3500 3000 2500 2000 1500 1000 500 0 0 100 200 300 400 Elution buffer % 100 149 mg 50 6.0 10.0 Elution buffer % 100 HisTrap FF 5 ml A280 mAU 4000 3500 3000 2500 2000 1500 1000 500 0 0.Histidine-tagged proteins Products featured: HisTrap FF columns. coli SDS-PAGE (Mr × 103) 97 Flow rates: 66 45 MBP-(His)6 System: 30 20 14 W ma rke Sta rs t ed rt m po ate ol His Elu ria Tra t ed l pF po F1 ol Elu His ml t ed Tra pF po ol F5 His ml Pre pF F1 6/1 0 Elu Summary LM .0 40.

Product Quantity Code No.com . Purification Detection Related products available by specific customer order For more information on Biacore systems and chromatography and/or electrophoresis systems.Histidine-tagged proteins Product Quantity Code No. please visit www.gelifesciences.

StrepStrep Strep Strep Strep Strep .

Multiphor II Electrophoresis System. coli Strep 2000 1500 A280 mAU GF step A280 mAU 70 60 50 40 1000 30 20 GF step: Column: Sample: Flow rate: Buffer: System: 500 10 0 0 10 20 30 40 ml 0 0 10 20 30 40 50 60 ml SDS-PAGE (Mr × 103) 97 66 45 30 20 14 PreScission (Histidine)6 tag mCherry MYG (85-87) Full-length target protein MYG (85-87) mCherry fragment TEV Strep-tag II TEV Strep-tag II PreScission (Histidine)6 tag mCherry fragment Summary Strep . HiLoad 16/60 Superdex 75 pg. ÄKTAprime plus. coli AC step: Column: Sample: Flow rate: Binding buffer: Elution buffer: AC step E. LMW-SDS Marker Kit Strep Strep E. ExcelGel SDS Gradient 8–18.Strep-tag II proteins Products featured: StrepTrap HP.

Strep-tag II proteins Products featured: StrepTrap HP. ÄKTAxpress Strep Strep Strep SDS-PAGE Column: Sample: Strep (Mr × 103) Binding buffer: Elution buffer: Flow rates: System: 188 98 62 49 38 28 17 14 60 30 rs h ate ug rke po ol (His)6-Streptag II-protein Lys hro Ma Flo Acknowledgements: Summary Elu t ed tar ge tp rot e wt in .

0 ml 0 0 50 100 150 ml 1000 1000 0 0 200 400 600 700 ml 1000 0 0 0 Columns: Sample: Sample volumes: Regeneration: Binding buffer: Elution buffer: Flow rates: Strep E. XK 26/20 column.0 20. ÄKTAexplorer Strep E.Strep-tag II proteins Products featured: StrepTrap HP columns. coli StrepTrap HP 1 ml A280 mAU 3000 = A 280 = A 587 = Elution buffer. StrepTactin Sepharose High Performance. coli System: Summary . %B StrepTrap HP 5 ml A587 mAU 3000 A280 mAU 3000 StrepTactin Sepharose High Performance XK 26/20 A587 mAU 3000 A280 mAU 3000 A587 mAU 3000 2000 2000 1000 2000 2000 2000 2000 1000 1000 0 0.0 10.

gelifesciences. please visit www.Strep-tag II proteins Product Quantity Code No. Purification Related products For more information on chromatography and/or electrophoresis systems.com .

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M MB dil LM le. LMW-SDS Marker Kit δ A280 mAU 2000 SDS-PAGE Eluted pool (Mr × 10 ) 3 1500 1000 500 97 66 45 30 0 10 20 30 40 ml MBP*2paramyosin-Sal 0 rke d1 Tra p PT rap ma ute W . ExcelGel SDS Gradient 8–18.MBP-tagged proteins Products featured: MBPTrap HP. coli HP :15 HP rs g . ÄKTAprime plus. :15 mp Sa d1 h. Multiphor II Electrophoresis System. 120 100 50 0 0 20 40 60 80 100 120 ml GF step: Column: Sample: Flow rate: Buffer: Summary Flo wt hro HiL ug oa A280 mAU ute dil dS up erd ex 16 Elution buffer: /60 BP 20 0p AC step: Column: Sample: Flow rate: Binding buffer: 20 14 δ E. HiLoad 16/60 Superdex 200 pg.

di p H lute s Elu d1 P. d ted :6 ilu fra ted cti 1:6 on sf rom MB PT rap HP Elu ted fra cti on sf rom Su pe rde x2 00 pg MBPMCAD AC step Column: Sample: Binding buffer: Elution buffer: Flow rate: System: E. coli A280 mAU 400 300 200 100 0 0 20 40 60 80 ml GF step Column: Sample: Buffer: Flow rate: System: Acknowledgements: Summary Flo .MBP-tagged proteins Products featured: MBPTrap HP. ÄKTAprime A280 mAU 2500 2000 1500 1000 500 0 0 20 40 60 80 100 120 ml SDS-PAGE (Mr × 103) 170 130 95 72 56 43 34 26 17 11 Mo lec u wt Sta lar w hro eig rt m ug h M ate ht m ria ark BP er Tra l. XK 16/20. Superdex 200 prep grade.

MBP-tagged proteins Products featured: MBPTrap HP. coli 140 160 180 ml SDS-PAGE (Mr × 103) 250 130 100 70 55 35 27 15 10 we Mo igh lec t m ula ark r er Eluted fractions from gel filtration Acknowledgements: Summary . ÄKTAxpress A280 mAU 2000 1500 1000 500 0 60 AC Elution buffer %B 100 80 60 40 20 0 80 100 120 GF AC column: Sample: Flow rate: Binding buffer: Elution buffer: GF column: Sample: Flow rate: Buffer: System: E. HiLoad 16/60 Superdex 200 pg.

0 15.d l. d ilu . Multiphor II Electrophoresis System. ÄKTAexplorer. Dextrin Sepharose High Performance. ExcelGel SDS Gradient 8–18.galactosidase 97 66 45 30 20 14 System: rke rs 1:6 2 2 1:3 1:1 ted 1:1 1:3 1:3 ted ate ria l .MBP-tagged proteins Products featured: MBPTrap HP columns.0 20. coli SDS-PAGE Eluted pool (Mr × 10 ) 3 Flowthrough Sample volumes: Binding buffer: Elution buffer: Flow rates: MBP2*.d ilu ted 1:3 . LMW-SDS Marker Kit β MBPTrap HP 1 ml A280 mAU 3000 2500 2000 1500 1000 500 0 0.d LM . d ilu l.0 5.0 ml 20 0 80 60 40 Elution Buffer % 100 Dextrin Sepharose High Performance XK 26/20 A280 mAU 3000 2500 2000 1500 40 1000 500 0 0 100 200 300 400 500 600 ml 20 0 80 60 Elution Buffer % 100 Columns: Sample: β E. d 1m 1m /20 5m ml HP 5 HP 26 HP HP XK 26 PT rap PT rap PT rap Tra p MB P MB MB Summary MB Sta rt m XK /20 . XK 26/20 column.d ilu ted ted ma ilu ted ilu ted ilu W l.0 10.

please visit www.com .gelifesciences. Purification Purification For more information on Biacore systems and chromatography and/or electrophoresis systems.MBP-tagged proteins Product Quantity Code No.

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ÄKTAxpress. LMW-SDS Marker Kit Run 1 Run 2 Run 3 HiPrep™ 16/10 HiPrep™ 16/10 HiPrep™ 16/10 HiPrep™ 16/10 HisTrap HP HiPrep 26/10 Desalting GSTrap 4B HiPrep 26/10 Desalting HisTrap HP HiPrep 26/10 Desalting GSTrap 4B HiPrep 26/10 Desalting SDS-PAGE Sample: Columns: E.coli (Mr × 10 ) 3 Affinity chromatography: Desalting: Buffers: 96 66 (His)6-GFP-GST 45 30 20 14 HisTrap HP Binding buffer: Elution buffer: GSTrap 4B: Binding buffer: Elution buffer: HiPrep 26/10 Desalting: Buffer: Summary W ma rke hro rs ug hr un Elu 1 tio nr Flo un wt 1 hro ug hr un 2 Elu tio nr Flo un wt 2 hro ug hr un 3 Elu tio nr un 3 Flo wt LM . ExcelGel SDS Gradient 8–18.Dual-tagged proteins Products featured: HisTrap HP. GSTrap 4B. Multiphor II Electrophoresis System.

StrepTrap HP. coli Individual HisTrap HP (Mr × 103) HisTrap HP + StrepTrap HP Individual StrepTrap HP Individual HisTrap HP purification Column: Binding buffer: Elution buffer: Flow rate: Individual StrepTrap HP purification Column: Binding buffer: Elution buffer: 188 98 62 49 38 28 17 14 6 3 r h ol gh ol ol rke ug po po po ug h wt hro u Dual-tagged Strep-tag II-(His)6 Flow rate: Two-step HisTrap HP and StrepTrap HP purification Column: Binding buffer: Elution buffer: Flow rate: Column: Sample: Binding buffer: Elution buffer: Flow rate: System: hro ma Elu ted wt ted Elu ed eig ht Acknowledgements: Mo lec Summary ula rw Flo wt Flo Flo Elu t hro .Dual-tagged proteins Products featured: HisTrap HP. coli SDS-PAGE Sample: Strep E. ÄKTAxpress Strep E.

ExcelGel SDS Gradient 8–18. HisTrap HP. Multiphor II Electrophoresis System. ÄKTAxpress.1 14.4 1000 500 0 70 80 90 100 110 Loop wash 120 130 140 ml HisTrap HP StrepTrap HP elution flow through StrepTrap HP elution A 280 mAU 700 600 500 400 300 200 100 0 70 StrepTrap HP elution Mr × 103 96 66 45 (His)6-mCherry-Strep-tag II 30 20.1 14.Dual-tagged proteins Products featured: StrepTrap HP. LMW-SDS Marker Kit Strep Columns: Sample: Strep E.4 80 HisTrap HP flow through 90 100 110 ml HisTrap HP elution Summary . coli Binding buffer (StrepTrap HP): A 280 mAU Mr × 103 2000 Elution buffer (StrepTrap HP): Binding buffer (HisTrap HP): Elution buffer (HisTrap HP): System: 1500 (His)6-mCherry-Strep-tag II 96 66 45 30 20.

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