Jessica Nguyen Glycoprotein Chromatography April 26, 2013

Partners: Tyler aclay, Gol!ner "igaro #$210%&01: 'r( )napp

Introduction:
Chromatography is a la*oratory techni+ue that separates certain components o, a mi-ture
,rom its surroun!ings through mo.ement o, a stationary phase( Chromatography is a use,ul
la*oratory techni+ue /here it can ta0e a!.antage o, the elements o, speci,icity o, certain
molecules that are also !epen!ent on charge an! acti.e sites as /ell(
1igan!s are sugar molecules that ha.e a ten!ency to *in! to speci,ic acti.e sites on
certain molecules in a cell( The *in!ing o, any ligan! may ha.e se.eral purposes such as
2ser.ing a *iological purpose to signal triggering molecular *in!ing sites on a target protein3
4#aron 20105( This interaction o, the *in!ing o, a protein an! a ligan! can *e manipulate! to
ser.e as a puri,ication process( $n serum, /hich is centri,ugation o, proteins ,rom *loo!, proteins
that ha.e certain ligan! speci,icity /ill *in! to only one speci,ic ligan!( There,ore, the
manipulation o, this *in!ing *et/een protein&ligan! comple-es can *e ta0en a!.antage o,
through means o, a,,inity chromatography(
The o*6ecti.e o, chromatography is to separate elements( Thin layer chromatography is a
separation techni+ue that is commonly use! in chemistry /hile a,,inity chromatography is
commonly use! in *iological e-perimentation( $n terms, o, chemistry, thin layer chromatography
is one o, the more pre,erre! metho!s o, separation !ue to the e,,ecti.eness an! cre!i*ility ,or the
natural mi-tures( There are !i,,erent mi-tures that tra.els an! mo.e at !i,,erent paces !epen!ing
on /hat solution is *eing use as the a!sor*ent an! as /ell as other !i,,erent partitions( The
components o, the mi-tures also ha.e an e,,ect on ho/ ,ast or slo/ the mi-ture /ill tra.el(
7eparation through the means o, chromatography *e may preparati.e or analytical( The /hole
purpose o, preparati.e chromatography is to separate se.eral components o, a mi-ture into larger
+uantities /ith large similarities( 8n the other han!, analytical chromatography is normally
associate! /ith smaller +uantities o, materials to !etermine relati.e proportions o, un0no/ns in
a mi-ture(
There are se.eral purposes o, thin layer chromatography that inclu!e the purposes to
esta*lish the num*er o, components in a mi-ture o, organic compoun!s, to esta*lish the i!entity
o, an un0no/n su*stance, to monitor the progress o, an organic reaction, to !etermine the
e,,ecti.eness o, a puri,ication, to !etermine the appropriate sol.ent system ,or column
chromatography, an! to monitor the column chromatographic separation o, organic compoun!s
*y testing ,ractions( 'ue to thin layer chromatography *eing a relia*le techni+ue ,or separation,
it is usually the pre,erre! metho! o, isolation o, a su*stance or se.eral un0no/n puri,ie!
su*stances ,or column chromatography an! gas chromatography /hich typically ,ollo/ a,ter( An
important ,actor o, thin layer chromatography is the sol.ent as /ell as the a!sor*ent( 7ilica gel,
cellulose, an! alumina are usually the coate! sur,aces o, chromatography plates( They are the
a!sor*ents on /hich the actual separation is carrie! out on the thin layer( The important ,actor o,
these a!sor*ents to *e relia*le separators is /hen the plate is actually place! in the sol.ent
system, the sol.ent ,ront /ill mo.e an! as it mo.es !i,,erent compoun!s mo.e to !i,,erent
e-tents on the sur,ace o, the plate !ue to continuous !i,,erential a*sorption an! !i,,erential
elution occurring *et/een the stationary an! mo*ile sol.ent phase( The polarity o, the sol.ent
system is one o, the many important ,actors that !etermine the e,,ecti.eness o, T1C as an
analytical tool(
Thin layer chromatography can *e use! as an analogy ,or a,,inity chromatography( The i!eal
mechanism o, a,,inity chromatography ,ollo/s the *in!ing an! charge nature o, thin layer
chromatography( A,,inity chromatography is more e,,ecti.e ,or *iological e-perimentation !ue
to the !i,,erence o, su*stances that are teste!( This metho! o, chromatography is e-tremely
success,ul /here it allo/s e-aminers to *e a*le to isolate certain large molecules such as
proteins ,rom a selection o, mi-tures o, proteins /ith little margin ,or error( The concept o, the
sol.ent phase ,or a,,inity chromatography is the i!ea *ehin! certain molecule *in!ing *ea!s that
are use! to *loc0 certain sol.ents( A,,inity chromatography ,ollo/s the mechanism that any
su*stance can *e isolate! *y running a *in!ing mo*ile structure ,or ligan!s to *in!( A,,inity
chromatography is a .ersatile metho! that is e-tremely e,,ecti.e an! is usually use! in the
process o, the puri,ication o, a protein&ligan! *in!ing comple-( The separation o, a ,e/
milligrams to a hun!re! grams can *e !one e,,iciently( The e,,iciency o, a,,inity
chromatography is !epen!ent on the sampling mo.ing !o/n a column continuously partitioning
occurs *et/een the mo.ing sol.ent ,rom the stationary a!sor*ent, /hich in this case are
concana.alin&A *ea!s(
The protein sample that is to *e separate! is applie! at the top o, the a,,inity column,
/here it *egins to 6ourney !o/n/ar!s through a column in a mo*ile phase( 7imilar to T1C, the
mo.ing sol.ent is .isi*ly mo.ing through the column an! !epen!ing on the *in!ing a,,inity o,
the *ea!s that are *eing use! as the a!sor*e! on the sur,ace o, the a!sor*ent to a !i,,erent e-tent(
The suita*le sol.ent /ith similar solu*le characteristics in this case mannose, can *e use! to
e-tract the *oun! protein ,rom the *in!ing sites o, the concana.alin *ea!s as an elutant that /ill
allo/ the separation at an e,,ecti.e an! constant rate( 8ne aspect o, a,,inity chromatography that
is essential is the su*6ection o, samples through electrophoresis( The gel that is use! !uring the
su*6ection to electrophoresis is 7'7( 7'7 is use! as a !enaturation agent that mas0s the nati.e
charges aroun! any proteins, an! pro.i!es all proteins /ith a negati.e charge( 'uring the
analysis o, samples that ha.e *een su*6ecte! to electrophoresis, the samples are purely tra.eling
*ase! on their molecular /eights( 'ue to the general process o, running electrophoresis an!
creating an electric ,iel! ,or the proteins to tra.el to a positi.e en!, /hich results in the hea.ier
protein comple-es to tra.el slo/er co.ering less !istance on the gel( This allo/s the analysis an!
!etermination o, /hich protein has *een isolate!( 9lectrophoresis su*6ection o, isolate! samples
are o,ten run alongsi!e sample proteins that ser.e as a control on a stan!ar! cur.e( :ith proteins
an! a stan!ar! cur.e o, molecular /eights, the un0no/n protein can *e i!enti,ie! through means
o, a semilog graph that in!icates the relationship o, proteins through molecular /eights an! the
!istance tra.ele! a,ter electrophoretic su*6ection(
;sing the principles o, chromatographic separation, a,,inity chromatography /as use! to
analy<e the o*6ecti.e o, the e-periment, /hich /as to !etermine /hich glycoproteins o, re!
*loo! cells contain glucose=mannose( Con&A *oun! to agarose *ea!s run a mi-ture o,
glycoproteins to see /hich contains glucose or mannose, /hich /ill *in! to Con&A, then run the
,lo/ through an! elutant through 7'7&chromatography against stan!ar! proteins( Their relati.e
/eights can then *e use! to i!enti,y /hich proteins contain mannose or glucose an! /hich
proteins !on>t(
Methods:
Reagents: Con&A *oun! a,,inity column 7'7&agarose gel
7tan!ar! Proteins 0(2 gCl
annose 49lutant5 Commasie #lue 7tain
9lectrophoresis *u,,er, 2- concentration Acetic Aci!
9-traction *u,,er 40(0? Tris&@Cl p@ A(35
Column *u,,er 40(1? NaCl, 10 m Tris p@ B(05
Research Design:
Modified from Poole and Hancock. l984. Eur.J.Biochem.44:!"#$!%&
$( Preparation o, sample
1( 8*taine! 2 plastic sample test tu*es an! ,ille! each /ith hal, o, !og *loo! sample(
2( 7u*6ecte! t/o test tu*es /ith !og *loo! samples ,or centri,ugation ,or 10 minutes at
?000 ra!=s to separate *loo! pellets ,rom sample(
$$( 8smotic shoc0 o, sample
1( Cesuspen!e! *loo! pellets, *oth test tu*es, ,rom centri,ugation in B(0m1 o, 0(0?
Tris&@Cl p@ A(3 an! 0(2 gCl(
2( $ncu*ate! ,or 10 minutes(
3( Chille! samples in a col! ice&/ater *ath ,or 1? minutes(
D( :arme! at 30
E
C ,or 10 minutes(
?( 7u*6ecte! C#C contents to ,urther centri,ugation to pellet out C#C contents(
$$$( Application o, samples to column /=mannose elution
1( Cun supernatant samples through ConA *ea!e! column(
2( Collect ,lo/ through ,raction(
3( :ash column /ith column *u,,er t/ice(
D( 9lute /ith mannose(
?( Collect elute! ,raction(
6( Prepare 20 F1 ,lo/ through ,raction /=electrophoresis *u,,er 420F m15
Prepare 20 F o, elute! ,raction /=electrophoresis *u,,er 420 F15
A( Cun prepare! samples /ith stan!ar! proteins(
$G( Application o, samples to column /=7'7 elution *u,,er
1( Cun supernatant samples through ConA *ea!e! column(
2( Collect ,lo/ through ,raction(
3( :ash column /ith column *u,,er t/ice(
D( 9lute /ith a,,inity chromatography 7'7 elution *u,,er
?( Collect elute! ,raction(
6( Prepare 20 F1 ,lo/ through ,raction /=electrophoresis *u,,er 420F m15
Prepare 20 F o, elute! ,raction /=electrophoresis *u,,er 420 F15
A( Cun prepare! samples /ith stan!ar! proteins(
Results:
"igure H1: 7'7 9lectrophoresis Gel Ta*le H1: 7'7 Gel )ey
Discussion:
The stan!ar! proteins run !i,,erent *an!s along the scale ,rom the electrophoresis
su*6ection( 7'7 PAG9 gel is responsi*le ,or mas0ing the nati.e charges o, proteins( These
charges are all gi.en an o.erall negati.e charge( The nature o, electrophoresis re+uires that
contents o, the /ells are su*6ecte! to a positi.e electrical ,iel!( This promotes the mo.ement o,
proteins purely through molecular /eight( The hea.ier a protein is the more li0ely it is to *e
locate! near the /ell origin( $n this la* e-periment, there /ere t/o trials to elute the proteins that
ha! a *in!ing a,,inity to concana.alin A *ea!s that /ere use! as a mo*ile phasing agent in the
column( The t/o .ariations *et/een the trials /as simply !uring the elution o, the *oun! protein
process( 8ne trial o, elute /as !one using mannose /hich *in!s to concana.alin A an! has a
high a,,inity( The mechanism *ehin! this *in!ing /as the reasoning that the proteins that
actually *oun! to the con&A *ea!s /oul! *e elute! /hile mannose /oul! *in! to the con&A
*ea!s( 7tan!ar! 7'7 elation *u,,er /as use! to elute proteins that *oun! to com&A( These t/o
metho!s are use! in comparison to one another to !etermine /hether glycoproteins in *loo!
serum coul! *e e,,ecti.ely isolate! through means o, a,,inity chromatography /ith con&A *ea!s(
This la* re+uire! the centri,ugation o, !og *loo! samples to smaller pellets( The
centri,ugation process separate! the plasma layer ,rom the *loo! pellet layer( The pellets /ere
too *ig to e.en put through the columns, there,ore they re+uire! ,urther centri,ugal ion as /ell as
shoc0ing the re! *loo! cell pellets to allo/ ,or the e-ploitation o, the glycoproteins /ithout
Well Sample
1 7tan!ar! Proteins
2 7upernatant
3 "lo/ Through "raction
4annose 9lutant5
D 9lute! "raction
4annose 9lutant5
? "lo/ Through "raction
47'7 9lution #u,,er5
6 9lute! "raction
47'7 9lution #u,,er5
completely !enaturing the proteins themsel.es( Pellets o, *loo! samples /ith proteins /ere
shoc0e! /ith a high concentration o, gCl to e-pose the proteins ,rom the normal nature(
"rom ,igure H1 /here /ell 3 an! /ell D in!icate the ,lo/ through ,ractions o, *oth the
7'7 elution *u,,er an! that ,rom the mannose elution processI the results sho/ .isi*le signs o,
proteins tra.eling se.eral !i,,erent !istances starting ,rom the origination o, the /ells( The ,lo/
through ,ractions containe! more proteins than that in the elute! ,raction *ecause there( 8nly
suppose! to *e a single one or t/o glycoproteins that coul! actually *in! to the con&A *ea!s(
"rom /ells HD an! H6, one can see that there are no protein *an!s( This in!icates that no proteins
that /ere *oun! to the com&A *ea!s /ere success,ully elute! ,rom the column( Although there
are no proteins in the elute! ,raction on the gel, the ,lo/ through ,ractions o, the gel present
in!icate that there /ere proteins that success,ully migrate! through the column( This sho/s that
there may not ha.e *een a high *in!ing a,,inity to the proteins( Although there /ere proteins that
ha.e a *in!ing nature to con&A *ea!s, the column *u,,er may ha.e ha! a stronger push to pull
the *ea!s through the ,lo/ through ,raction /hich /oul! lea.e nothing ,or the elution ,raction
a,ter/ar!s(
The supernatant /as run along the samples o, ,lo/ through ,raction o, *oth trials as a
control ,or /hat proteins /ere in the !og *loo! samples( 'uring the elution processes o, the
e-periment, the results !i! not support the hypothesis as an in!icate! result that the proce!ure /oul!
success,ully isolate the proteins that suppose!ly *oun! to the con&A *ea!s( This may ha.e *een a ,actor o, a
,e/ sources o, error, such as running the supernatant through the column, osmotic shoc0ing o, the the re!
*loo! cell samples as /ell as the preparing o, the samples ,or electrophoresis su*6ection( :hile the samples
/ere intro!uce! to the column, there /ere se.eral /ashe! to pre.ent any o, the supernatant an! the impurities
o, the samples( 'uring the /ashes, the column ha! a separate! layer an! 6ust the *ea!e! layer( The elution
*u,,er /as a!!e! in *oth situations o, mannose elute not an! 7'7 elution *u,,er, there /as a small ring o, re!
that tra.ele! 6ust *elo/ the line o, the con6ugate! con&A *ea!s( This can possi*ly *e conclu!e! as the proteins
that /ere *oun! to the *ea!s( #ecause the re! *loo! cell pellets are too *ig e.en a,ter centri,ugation, to tra.el
through the column(
'ue to the large nature o, the pellets, it /as not possi*le ,or the pellets to actually pass through the
con&A *ea!s that /ere so tightly pac0e! together( "rom the 7'7 gel selection, one coul! see that there /ere no
protein *an!s in either o, the elute! ,raction /ells /hich sho/s that no proteins /ere success,ully elute!
!uring the process( 8ne research aspect that this spar0e! /as ho/ one can e,,iciently isolate proteins using
chromatography techni+ues( Another +uestion that /as !irectly ,ormulate! ,rom the proce!ure propose! in the
e-periment is ho/ can one success,ully pellet the re! *loo! cell pellets to *e small enough to go through the
con&A columns(