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DNA Standards

Reference Guide
Welcome to the Little Book of
Standards: DNA Standards
This handbook is a practical guide to Bio-Rad’s complete line of DNA and protein
standards for electrophoresis and blotting. Bio-Rad standards are an excellent
means of monitoring electrophoresis and blotting experiments, or determining DNA
and protein sizes.

This handbook is organized into five sections for both DNA and protein standards.
On this side of the booklet, DNA standards are divided into sections based on
logical groupings of size ranges in base pairs (bp). On the other side, protein
standards are organized by general application.

Each page in this reference guide offers specifications for each set of standards,
recommended applications, a photograph of the standards run on a gel, and the
length in bp or the mass of each standard band. Each section divider contains
gel migration summaries for the standards described in that section.

We hope you find this booklet to be a useful guide to the selection and use of
Bio-Rad DNA and protein standards. For more information on products or
applications, contact your local Bio-Rad representative, or visit us on the Web
at discover.bio-rad.com

1
Bio-Rad DNA Standards and
Molecular Mass Markers
20 bp–2 kb DNA Standards 3–8 5 kb–5.7 Mb DNA Standards 22–28
20 bp Molecular Ruler 4 CHEF DNA Size Standards, 5 kb Ladder 23
EZ Load™ 20 bp Molecular Ruler 5 CHEF DNA Size Standards, 8 – 48 kb Ladder 24
AmpliSize® Molecular Ruler 6 CHEF DNA Size Standards, Lambda Ladder 25
100 bp Molecular Ruler 7 CHEF DNA Size Markers, 26
EZ Load 100 bp Molecular Ruler 8 S. cerevisiae Chromosomes
CHEF DNA Size Markers, 27
100 bp–10 kb DNA Standards 9 –17 H. wingei Chromosomes
EZ Load™ HT 100 bp –2 kb Molecular Marker 10 CHEF DNA Size Markers, 28
EZ Load HT 500 bp–10 kb Molecular Marker 11 S. pombe Chromosomes
100 bp PCR Molecular Ruler 12
Fluorescent DNA Standards 29–31
EZ Load 100 bp PCR Molecular Ruler 13 100 bp Fluorescein-Labeled Ruler 30
500 bp Molecular Ruler 14 100 bp Texas Red-Labeled Ruler 31
EZ Load 500 bp Molecular Ruler 15
Precision Molecular Mass Ruler 16
EZ Load Precision Molecular Mass Ruler 17

1–35 kb DNA Standards 18– 21


1 kb Molecular Ruler 19
EZ Load 1 kb Molecular Ruler 20
2.5 kb Molecular Ruler 21

2
20 bp–2 kb DNA Standards
Bio-Rad molecular rulers are available in several convenient size ranges and
increments for sizing single- and double-stranded DNA. Visually distinct reference
bands make it easy to determine the size of your sample DNA.

2,000 bp
1,500
1 kb
800 bp 800 bp
700
500 500 500

400 400 400

300 300 300

200 200 200

100 bp

60
50
40

20

10 bp

20 bp 100 bp AmpliSize®
Molecular Molecular Molecular

DNA Standards
Ruler Ruler Ruler

20 bp–2 kb
3
20 bp Molecular Ruler
Size Range Quantity Recommended Load Volume

20–1,000 bp, 50 µg DNA in 250 µl 2.5 µl (~500 ng DNA)


50 bands in exact TE buffer, pH 8.0
20 bp increments (0.2 µg/µl DNA)

The perfect 50% GC content of Bio-Rad’s precision-


1,0ˇ00 bp
sized molecular rulers eliminates spurious migration
due to sequence variation, ensuring that the fragments
migrate exactly according to their specified size.
500
Recommended gels: 1.5–2.5% standard agarose,
2.5 – 4% Certified™ low range ultra agarose, or 8 –12%
polyacrylamide.

Note: Add any conventional sample loading buffer


Reference 200
prior to loading. Store at 4°C. band 180
160
Catalog #: 170-8201 140
120
100
80

60

40

20

4
EZ Load™ 20 bp Molecular Ruler
Size Range Quantity Recommended Load Volume

20 –1,000 bp, 50 µg DNA in 500 µl 5 µl (~500 ng DNA)


50 bands in exact sample loading buffer
20 bp increments (0.1 µg/µl DNA)

EZ Load molecular rulers are precision-sized DNA


1,000 bp
fragments blended with sample loading buffer for
convenience. The perfect 50% GC content eliminates
spurious migration due to sequence variation,
ensuring that the fragments migrate exactly according 500

to their specified size.

Recommended gels: 1.5–2.5% standard agarose,


2.5 – 4% Certified™ low range ultra agarose, or 8 –12%
polyacrylamide. Reference 200
band 180
160
Note: Premixed in sample loading buffer containing 140
5% glycerol, 15 mM Tris, pH 8.0, 1.5 mM EDTA, 120

0.04% Bromophenol Blue, 0.04% Xylene Cyanole FF. 100


80
Store at 4°C.
60
Catalog #: 170-8351
40

20

5
®
AmpliSize Molecular Ruler
Size Range Quantity Recommended Load Volume

50 –2,000 bp, 25 µg DNA in 250 µl 5 µl (~50 ng DNA/band)


10 bands of proprietary TE buffer, pH 8.0
blunt-ended DNA (10 ng/band/µl)

Ten fragments of proprietary blunt-ended DNA of


2,000 bp
precise length and known sequence. The perfect 50%
1,500
GC content eliminates spurious migration due to
sequence variation, ensuring that the fragments 1,000
migrate exactly according to their specified size.
700
Recommended gels: 1.0–2.5% standard agarose,
2– 4% Certified™ low range ultra agarose, or 5–10% 500

polyacrylamide. 400

Note: Add any conventional sample loading buffer 300


prior to loading. Store at 4°C.

Catalog #: 170-8200 200

100

50

6
100 bp Molecular Ruler
Size Range Quantity Recommended Load Volume

100 –1,000 bp, 25 µg DNA in 250 µl 2.5 µl (~250 ng DNA)


10 bands in exact TE buffer, pH 8.0
100 bp increments (0.1 µg/µl DNA)

The perfect 50% GC content of this precision-sized


molecular ruler eliminates spurious migration due to 1,000 bp

sequence variation, ensuring that the fragments


migrate exactly according to their specified size.

Recommended gels: 1.0–2.5% standard agarose,


2– 4% Certified™ low range ultra agarose, or 5–10% 500
polyacrylamide.

Note: Add any conventional sample loading buffer


prior to loading. The 100 bp ruler may show a double-
or triple-banding pattern in polyacrylamide gels. The
DNA fragments in this product have HindIII compatible
cohesive ends. Store at 4°C.

Catalog #: 170-8202

100

7
EZ Load™ 100 bp Molecular Ruler
Size Range Quantity Recommended Load Volume

100 –1,000 bp, 25 µg DNA in 500 µl 5 µl (~250 ng DNA)


10 bands in exact sample loading buffer
100 bp increments (0.05 µg/µl DNA)

EZ Load molecular rulers are precision-sized DNA


fragments blended with sample loading buffer for 1,000 bp

convenience. The perfect 50% GC content eliminates


spurious migration due to sequence variation,
ensuring that the fragments migrate exactly according
to their specified size.
500
Recommended gels: 1–2.5% standard agarose or
5 – 8% polyacrylamide.

Note: Premixed in sample loading buffer containing


5% glycerol, 15 mM Tris, pH 8.0, 1.5 mM EDTA,
0.04% Bromophenol Blue, 0.04% Xylene Cyanole FF.
The 100 bp ruler may show a double- or triple-
banding pattern in polyacrylamide gels. The DNA
fragments in this product have HindIII compatible
cohesive ends. Store at 4°C.

Catalog #: 170-8352
100

8
100 bp–10 kb DNA Standards
Bio-Rad molecular rulers are available in several convenient size ranges and
increments for sizing single- and double-stranded DNA. Visually distinct reference
bands make it easy to determine the size of your sample DNA.

10 kb
10,000 bp
8,000 bp

5,000
4,000

3,000 bp

2,000 bp 2,000 2,000

1 kb
1,000 1,000 1,000 bp (100 ng)
700 (70 ng)
500 500 500 500 500 (50 ng)

DNA Standards
400

100 bp– 10 kb
300

200 200 200 (20 ng)

100 bp
100 100 bp (10 ng)

EZ Load™ HT EZ Load HT 100 bp 500 bp Precision


100 bp–2 kb 500 bp–10 kb PCR Molecular Molecular
Molecular Molecular Molecular Ruler Mass Ruler
Marker Marker Ruler

9
EZ Load™ HT 100 bp–2 kb
Molecular Marker
Size Range Quantity Recommended Load Volume

100–2,000 bp, 16 µg DNA in 1.6 ml 10 µl (~100 ng DNA)


5 bands that resolve TE buffer, pH 8.0
sharply in a short (~2 cm) (10 µg/ml DNA)
migration distance

EZ Load HT molecular markers are designed for 2,000 bp


use in gels with a short migration distance, such as 1,000
ReadyAgarose™ 96 Plus precast gels. These are
500
prediluted, ready-to-load molecular markers with
Orange G tracking dye to monitor electrophoresis.
200
Recommended gels: 3% ReadyAgarose 96 Plus
precast gel; 3.0 – 4.0% standard agarose. 100

Catalog #: 170-8361

10
EZ Load™ HT 500 bp–10 kb
Molecular Marker
Size Range Quantity Recommended Load Volume

500–10,000 bp, 16 µg of DNA in 1.6 ml 10 µl (~100 ng DNA)


5 bands that resolve TE buffer, pH 8.0
sharply in a short (~2 cm) (10 µg/ml DNA)
migration distance

EZ Load HT molecular markers are designed for 10,000 bp


use in gels with a short migration distance, such as 4,000
ReadyAgarose™ 96 Plus precast gels. These are
2,000
prediluted, ready-to-load molecular markers with
Orange G tracking dye to monitor electrophoresis. 1,000

500
Recommended gels: 1% ReadyAgarose 96 Plus
precast gel; 0.8 –1.5% standard agarose.

Catalog #: 170-8362

11
100 bp PCR Molecular Ruler
Size Range Quantity Recommended Load Volume

100 – 3,000 bp, 40 µg DNA in 200 µl 2 µl (~400 ng DNA)


30 bands in exact TE buffer, pH 8.0
100 bp increments (0.2 µg/µl DNA)

The perfect 50% GC content of this precision-sized


Reference 3,000 bp
molecular ruler eliminates spurious migration due to band
sequence variation, ensuring that the fragments
migrate exactly according to their specified size.
2,000
Recommended gels: 0.8 –2% standard agarose or
5% polyacrylamide.

Note: Add any conventional sample loading buffer


prior to loading. The DNA fragments in this product
have HindIII compatible cohesive ends. Reference 1,000
band
Store at 4°C.

Catalog #: 170-8206

500

100

12
EZ Load™ 100 bp
PCR Molecular Ruler
Size Range Quantity Recommended Load Volume

100 – 3,000 bp, 40 µg DNA in 500 µl 5 µl (~400 ng DNA)


30 bands in exact sample loading buffer
100 bp increments (0.08 µg/µl DNA)

EZ Load molecular rulers are precision-sized DNA


Reference 3,000 bp
fragments blended with sample loading buffer for band
convenience. The perfect 50% GC content eliminates
spurious migration due to sequence variation,
ensuring that the fragments migrate exactly according 2,000

to their specified size.

Recommended gels: 0.8 –2.0% standard agarose


or 5% polyacrylamide.

Note: Premixed in sample loading buffer containing Reference 1,000


band
5% glycerol, 15 mM Tris, pH 8.0, 1.5 mM EDTA,
0.04% Bromophenol Blue, 0.04% Xylene Cyanole FF.
Store at 4°C.
500
Catalog #: 170-8353

100

13
500 bp Molecular Ruler
Size Range Quantity Recommended Load Volume

500 – 8,000 bp, 40 µg DNA in 200 µl 2 µl (~400 ng DNA)


16 bands in exact TE buffer, pH 8.0
500 bp increments (0.2 µg/µl DNA)

The perfect 50% GC content of this precision-sized


molecular ruler eliminates spurious migration due to 8,000 bp
sequence variation, ensuring that the fragments
migrate exactly according to their specified size.
Reference 5,000
band
Recommended gel: 1.0 –1.5% standard agarose. 4,000

Note: Add any conventional sample loading buffer 3,000


prior to loading. The DNA fragments in this product
have EcoRI compatible cohesive ends.
Store at 4°C. 2,000

Catalog #: 170-8203

1,000

500

14
EZ Load™ 500 bp Molecular Ruler
Size Range Quantity Recommended Load Volume

500 – 8,000 bp, 40 µg DNA in 500 µl 5 µl (~400 ng DNA)


16 bands in exact sample loading buffer
500 bp increments (0.08 µg/µl DNA)

EZ Load molecular rulers are precision-sized DNA


fragments blended with sample loading buffer for 8,000 bp
convenience. The perfect 50% GC content eliminates
spurious migration due to sequence variation,
Reference 5,000
ensuring that the fragments migrate exactly according band
4,000
to their specified size.

Recommended gels: 1.0 –1.5% standard agarose. 3,000

Note: Premixed in sample loading buffer containing


5% glycerol, 15 mM Tris, pH 8.0, 1.5 mM EDTA, 2,000
0.04% Bromophenol Blue, 0.04% Xylene Cyanole FF.
The DNA fragments in this product have EcoRI
compatible cohesive ends. Store at 4°C.

Catalog #: 170-8354
1,000

500

15
Precision Molecular Mass Ruler
Size Range Quantity Recommended Load Volume

100 –1,000 bp 25 µg DNA in 250 µl 2.5 µl (~250 ng)


Mass Range TE buffer, pH 8.0
10 –100 ng (±1%) (0.1 µg/µl DNA)

The precision molecular mass ruler allows accurate


quantitation of the amount of DNA in a sample band.

Recommended gels: 1–2% standard agarose,


2– 4% Certified™ low range ultra agarose, or 5–8% 1,000 bp (100 ng)
polyacrylamide.
700 (70 ng)
Note: Add any conventional sample loading buffer
prior to loading. To maintain accurate fragment 500 (50 ng)
concentrations, spin down any condensate and mix
thoroughly prior to opening tube. The DNA fragments
in this product have either EcoRI or HindIII compatible 200 (20 ng)

cohesive ends. Store at 4°C. 100 (10 ng)

Catalog #: 170-8207

16
EZ Load™ Precision
Molecular Mass Ruler
Size Range Quantity Recommended Load Volume

100 –1,000 bp 25 µg DNA in 500 µl 5 µl (~250 ng)


Mass Range sample loading buffer
10 –100 ng (±1%) (0.05 µg/µl DNA)

The EZ Load precision molecular mass ruler allows


accurate quantitation of the amount of DNA in a
sample band. The ruler has been blended with sample
loading buffer for convenience.
1,000 bp (100 ng)
Recommended gels: 1–2% standard agarose,
2– 4% Certified™ low range ultra agarose, or 5–8% 700 (70 ng)
polyacrylamide.
500 (50 ng)
Note: Premixed in sample loading buffer containing
5% glycerol, 15 mM Tris, pH 8.0, 1.5 mM EDTA,
0.04% Bromophenol Blue, 0.04% Xylene Cyanole FF. 200 (20 ng)

To maintain accurate fragment concentrations, spin 100 (10 ng)

down any condensate and mix thoroughly prior to


opening tube. The DNA fragments in this product
have either EcoRI or HindIII compatible cohesive ends.
Store at 4°C.

Catalog #: 170-8356

17
1–35 kb DNA Standards
Bio-Rad molecular rulers are available in several convenient size ranges and
increments for sizing single- and double-stranded DNA. Visually distinct reference
bands make it easy to determine the size of your sample DNA.

35.0 kb
30.0

DNA Standards
25.0

1– 35 kb
20.0
15.0 kb 15.0
10 kb 12.5

7.0 7.5
6.0
5.0 5.0

4.0

3.0
2.5
2.0

1 kb

1 kb 2.5 kb
Molecular Molecular
Ruler Ruler

18
1 kb Molecular Ruler
Size Range Quantity Recommended Load Volume

1–15 kb, 40 µg DNA in 200 µl 2 µl (~400 ng DNA)


15 bands in exact TE buffer, pH 8.0
1 kb increments (0.2 µg/µl DNA)

The perfect 50% GC content of this precision-sized


molecular ruler eliminates spurious migration due to
sequence variation, ensuring that the fragments 15 kb

migrate exactly according to their specified size. 10

Recommended gel: 0.7–1.5% standard agarose.


7
Note: Add any conventional sample loading buffer
prior to loading. The DNA fragments in this product Reference 5
band
have EcoRI compatible cohesive ends. Store at 4°C. 4

Catalog #: 170-8204
3

19
EZ Load™ 1 kb Molecular Ruler
Size Range Quantity Recommended Load Volume

1.0 –15 kb, 40 µg DNA in 500 µl 5 µl (~400 ng DNA)


15 bands in exact sample loading buffer
1.0 kb increments (0.08 µg/µl DNA)

EZ Load molecular rulers are precision-sized DNA


fragments blended with sample loading buffer for
convenience. The perfect 50% GC content eliminates 15 kb

spurious migration due to sequence variation, 10


ensuring that the fragments migrate exactly according
to their specified size. 7

Recommended gel: 0.7–1.5% standard agarose.


Reference 5
band
Note: Premixed in sample loading buffer containing 4
5% glycerol, 15 mM Tris, pH 8.0, 1.5 mM EDTA,
0.04% Bromophenol Blue, 0.04% Xylene Cyanole FF. 3
The DNA fragments in this product have EcoRI
compatible cohesive ends. Store at 4°C.
2
Catalog #: 170-8355

20
2.5 kb Molecular Ruler
Size Range Quantity Recommended Load Volume

2.5 –35 kb, 40 µg DNA in 400 µl 4 µl (~400 ng DNA)


14 bands in exact TE buffer, pH 8.0
2.5 kb increments (0.1 µg/µl DNA)

The perfect 50% GC content of this precision-sized


molecular ruler eliminates spurious migration due to
sequence variation, ensuring that the fragments 35 kb
30
migrate exactly according to their specified size. 25

Recommended gels: 0.7–1.0% standard agarose 20

for conventional electrophoresis, or 1.0 –1.5% for 15


pulsed field electrophoresis. 12.5
Reference 10
Note: Add any conventional sample loading buffer band
7.5
prior to loading. The DNA fragments in this product
have EcoRI compatible cohesive ends. Store at 4°C.
5.0

Catalog #: 170-8205

2.5

21
5 kb–5.7 Mb (Pulsed Field)
DNA Standards

DNA Standards
Bio-Rad offers several standards for DNA separations on pulsed field gels,

5 kb– 5.7 Mb
ranging from cosmid inserts to whole chromosomes.

5.7 Mb
4.6

3.13 Mb 3.5

2,200 kb
1,600 2.70
2.35
1.81
1.66
1,125 1.37
1 Mb 1,020 1.05
945

825
785
750
680
610
565
450
365
285
225

100 kb 145.5 kb
97.0
48.5

48.5 kb

38.4
33.5
29.9

24.8
22.6
19.4
17.1
14.7 kb 15.0
10 kb 12.2
10.0
9.8 8.6
8.3

4.9

5 kb 8–48 kb Lambda S. cerevisiae H. wingei S. pombe


Ladder Ladder Ladder Chromosomes Chromosomes Chromosomes
22
CHEF DNA Size Standards,
5 kb Ladder
Size Range Quantity Recommended Load Volume

4.9 –98 kb in 4.9 kb 20 µg DNA in 200 µl 8 –10 µl (0.8 –1 µg DNA)


increments 28 mM Tris, 50 mM
(concatemers EDTA, 28 mM NaCl,
of pBR328) 0.05% SDS, pH 8.0
(0.1 µg/µl DNA)

CHEF DNA size standards are ideally suited for size


estimation of larger DNA fragments separated by
pulsed field electrophoresis.

Recommended gel: 1.0 –1.5% standard agarose.

Note: Add a sample loading buffer prior to loading.


Store at 4°C.

Catalog #: 170-3624

14.7 kb

9.8

4.9

23
CHEF DNA Size Standards,
8–48 kb Ladder
Size Range Quantity Recommended Load Volume

8.3 – 48.5 kb (13 bands 25 µg DNA in 125 µl 1–5 µl (0.2 –1 µg DNA)


derived from DNA TE buffer, pH 8.0
digested with five (0.2 µg/µl DNA)
restriction enzymes)

CHEF DNA size standards are ideally suited for size


estimation of larger DNA fragments separated by 48.5 kb

pulsed field electrophoresis.

Recommended gel: 1.0 –1.5% standard agarose. 38.4

Note: Add a sample loading buffer and heat sample


33.5
at 65°C for 5 min before loading. Store at 4°C.
29.9
Catalog #: 170-3707
24.8
22.6

19.4
17.1
15.0
12.2
10.0
8.6
8.3

24
CHEF DNA Size Standards,
Lambda Ladder
Size Range Quantity Recommended Load Volume

48.5 – ~1,000 kb in 5 low-melt agarose See below


48.5 kb increments blocks, ~15 µg
(concatemers of lambda DNA per block
cl857Sam7)

CHEF DNA size standards are ideally suited for size


estimation of larger DNA fragments separated by
pulsed field electrophoresis.

Recommended gel: 0.8 –1.0% standard agarose.

Note: Cut a plug from a block into a size that will fit
a well of your gel (5 – 8 plugs per block). The height
of the plug should not extend above the well. After
placing the plug into a well, seal the well with melted
1.0% low-melt agarose and allow it to solidify. Store
blocks at 4°C.

Catalog #: 170-3635

145.5 kb
97
48.5

25
CHEF DNA Size Markers,
S. cerevisiae Chromosomes
Size Range Contents Recommended Load Volume

0.2–2.2 Mb 5 low-melt agarose See below


(16 chromosomes blocks containing
that are resolved into Saccharomyces
15 bands) cerevisiae chromosomes

CHEF DNA size standards are ideally suited for size


estimation of larger DNA fragments separated by
pulsed field electrophoresis. 2.2 Mb
1.6
Recommended gel: 0.7–1.0% standard agarose.

Note: Cut a plug from a block into a size that will fit 1.13
a well of your gel (5– 8 plugs per block). The height 1.0
0.95
of the plug should not extend above the well. After
0.83
placing the plug into a well, seal the well with melted 0.79
1.0% low-melt agarose and allow it to solidify. Store 0.75

blocks at 4°C. 0.68


0.61
0.57
Catalog #: 170-3605
0.45
0.37
0.29
0.225

26
CHEF DNA Size Markers,
H. wingei Chromosomes
Size Range Contents Recommended Load Volume

1– 3.1 Mb 5 low-melt agarose See below


(7 chromosomes) blocks containing
Hansenula wingei
chromosomes

CHEF DNA size standards are ideally suited for size


estimation of larger DNA fragments separated by
pulsed field electrophoresis.

Recommended gel: 0.7–1.0% standard or


chromosomal grade agarose. 3.13 Mb

Note: Cut a plug from a block into a size that will fit
a well of your gel (5– 8 plugs per block). The height of
the plug should not extend above the well. After
placing the plug into a well, seal the well with melted
1.0% low-melt agarose and allow it to solidify. Store
2.70
blocks at 4°C.
2.35
Catalog #: 170-3667

1.81
1.66

1.37

1.05

27
CHEF DNA Size Markers,
S. pombe Chromosomes
Size Range Contents Recommended Load Volume

3.5 – 5.7 Mb 5 low-melt agarose See below


(3 chromosomes) blocks containing
Schizosaccharomyces
pombe chromosomes

CHEF DNA size standards are ideally suited for size


estimation of larger DNA fragments separated by
pulsed field electrophoresis.
5.7 Mb
Recommended gel: 0.6 –1.0% standard or (Chromosome I)
chromosomal grade agarose.
4.6
(Chromosome lI)
Note: Cut a plug from a block into a size that will fit
a well of your gel (5– 8 plugs per block). The height of
the plug should not extend above the well. After
3.5
placing the plug into a well, seal the well with melted (Chromosome III)

1.0% low-melt agarose and allow it to solidify. Store


blocks at 4°C.

Catalog #: 170-3633

28
DNA Standards
Fluorescent
Fluorescent DNA Standards
Bio-Rad fluorescent DNA standards are ideal for fast, accurate imaging.
A choice of two fluorescent dyes provides multiplexing capabilities.

10 kb

1 kb
800 bp 800 bp

500 500

400 400

300 300

200 200

100 bp

100 bp 100 bp
Fluorescein- Texas Red-
Labeled Labeled
Ruler Ruler

29
100 bp Fluorescein-Labeled Ruler
Size Range Quantity Recommended Load Volume

100 –1,000 bp, 20 µg DNA in 100 µl 2 µl (~400 ng DNA)


10 bands in exact TE buffer, pH 8.0
100 bp increments (0.2 µg/µl DNA)

Fluorescently labeled standards are ideal for fast,


accurate imaging and precise, multiplex DNA sizing.
1,000 bp
Recommended gel: 1.0 –2.5% standard agarose or
5 –10% polyacrylamide. Bio-Rad’s Certified™ molecular 700

biology agarose minimizes the background effects


associated with endogenous agarose fluorescence. 500

Note: To increase sample throughput and accurately 400

determine fragment size and location, fluorescein


standards can be run in the same well (multiplexed) 300

with a sample or another standard containing a


complementary fluorescent label such as Texas Red.
Add any conventional sample loading buffer prior to 200
loading.

Excitation: 302 and 494 nm

Emission maximum: 520 nm. Store in the dark


at 4°C. 100

Catalog #: 170-8216

30
100 bp Texas Red-Labeled Ruler
Size Range Quantity Recommended Load Volume

100 –1,000 bp, 20 µg DNA in 100 µl 2 µl (~400 ng DNA)


10 bands in exact TE buffer, pH 8.0
100 bp increments (0.2 µg/µl DNA)

Fluorescently labeled standards are ideal for fast,


accurate imaging and precise, multiplex DNA sizing.
1,000 bp
Recommended gel: 1.0 –2.5% standard agarose or
5 –10% polyacrylamide. Bio-Rad’s Certified™ molecular 700

biology agarose minimizes the background effects


associated with endogenous agarose fluorescence. 500

Note: To increase sample throughput and accurately 400

determine fragment size and location, Texas Red


standards can be run in the same well (multiplexed) 300

with a sample or another standard containing a


complementary fluorescent label such as fluorescein.
Add any conventional sample loading buffer prior to 200
loading.

Excitation: 302 and 596 nm

Emission maximum: 615 nm. Store in the dark


at 4°C. 100

Catalog #: 170-8217

31
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Protein Standards
Reference Guide
Welcome to the Little Book of
Standards: Protein Standards
This handbook is a practical guide to Bio-Rad’s complete line of protein and
DNA standards for electrophoresis and blotting. Bio-Rad standards are an
excellent means of monitoring electrophoresis and blotting experiments, or
determining protein and DNA sizes.

This handbook is organized into five sections for both protein and DNA standards.
On this side of the booklet, protein standards are divided into sections based
on applications. Featured are our new Precision Plus Protein™ standards,
highly purified recombinant proteins that offer a new level of band clarity and
application versatility.

Each page in this reference guide offers specifications for each set of standards,
recommended applications, a photograph of the standards run on a gel, and the
molecular mass or pI of each standard band. Each section divider contains gel
migration summaries for the sets of standards described in that section.

We hope you find this booklet to be a useful guide to the selection and use
of Bio-Rad protein and DNA standards. For more information on products or
applications, contact your local Bio-Rad representative, or visit us on the Web
at discover.bio-rad.com

1
Bio-Rad Protein Molecular Weight
and Reference Standards
Precision Plus Protein™ Standards 3 –7 Blotting Standards 22– 25
Precision Plus Protein Standards, Unstained 5 Biotinylated SDS-PAGE Standards, 23
Precision Plus Protein Standards, All Blue 6 Broad Range

Precision Plus Protein Standards, Dual Color 7 Biotinylated SDS-PAGE Standards, 24


Low Range
Unstained Standards 8 –15 Biotinylated SDS-PAGE Standards, 25
SDS-PAGE Standards, Broad Range 9 High Range
SDS-PAGE Standards, Low Range 10
2-D and IEF Standards 26– 28
SDS-PAGE Standards, High Range 11 2-D SDS-PAGE Standards 27
Polypeptide SDS-PAGE Standards 12 IEF Standards 28
Silver Stain SDS-PAGE Standards, 13
Low Range Protein Gel Stains 29
Silver Stain SDS-PAGE Standards, 14
High Range Molecular Weight Determination 30

SDS-PAGE Standards for SYPRO Orange, 15


Broad Range
Prestained Standards 16 – 21
Prestained SDS-PAGE Standards, 17
Broad Range
Prestained SDS-PAGE Standards, 18
Low Range
Prestained SDS-PAGE Standards, 19
High Range
Kaleidoscope™ Prestained Standards 20
Kaleidoscope Polypeptide Standards 21

2
Precision Plus Protein™ Standards
Protein Migration Chart

Migration of Precision Plus Protein standards on Criterion™ Tris-HCl pH 8.8 gels.


Select the acrylamide percentage that best resolves your protein or peptide of
interest in Laemmli gel buffered systems.
5% 7.5% 10% 12.5% 15% 18% 4–15% 4–20% 8–16% 10–20% 10.5–14%

250
250 150 250
250 150 100
250 100 75 250 250 150
150 150
100 75 50 250 100 150 100
250 150 250
37 100
75 75
50 150 75 75
100
150 150 50
250 37 25 100 50
75 50 37 50
20 100 75 37
100
37
75 50 37
25 15 25
50 37 25
150 75 20 50 20 20
10 25
37 25 37 15 25
20
15
20 15 15 10 20
100 50 25
20 10
10 15
15 10 15
37 10 10 10

Protein Standards
75 25

Precision Plus
Tris-HCI Gels

Protein molecular mass, kD. Migrations based on running gels until the dye front reached the bottom
of the gel.

3
Precision Plus Protein™ Standards
Protein Migration Chart

Migration of Precision Plus Protein standards on Criterion™ XT gels. Select the


acrylamide percentage that best resolves your protein or peptide of interest in
neutral pH gel buffered systems.
10% 12% 4–12% 10% 12% 4–12% 7% 3–8%

250
250 250 250 150
150 150 250 250 250
150 100
100 100
150 150
75 75 150 250
75 100
50 100
50 75 75 50 100 100
37 150
37 75
50 37
25 75
37 50 100
25 20 50
20 15 25 37 75
37 25
15 20 50
20
10 15
25 50
10 20 37
10 25 15
37
20 15
10

Criterion XT Bis-Tris gels Criterion XT Bis-Tris gels Criterion XT Tris-acetate


with XT MES running buffer: with XT MOPS running buffer: gels with XT Tricine
ideal for small proteins ideal for mid-size proteins running buffer:
ideal for large proteins

Protein molecular mass, kD. Migrations based on running gels until the dye front reached
the bottom of the gel.
4
Precision Plus Protein™ Standards,
Unstained
Size Range Quantity Recommended Load Volume

10 proteins, 10 –250 kD 360 µg protein in 1,000 µl Mini gels: 10 µl


of 30% (v/v) glycerol, (Coomassie R-250 stain);
2% SDS, 62.5 mM Tris, 1– 6 µl (silver stain)
pH 6.8, 50 mM DTT, Large gels: 20 µl
5 mM EDTA, 0.02% NaN3, (Coomassie R-250 stain);
0.01% Bromophenol Blue 3–12 µl (silver stain)

For accurate molecular weight determinations on 250 kD


an SDS-polyacrylamide gel or immunoblot.
150
Recommended gel: 4–20% gradient gel. See
protein migration chart on page 3 for approximate 100
migration distances with various gel percentages.
75

Note: Standards are premixed with sample


loading buffer. No dilution or heating is required.* 50

Contain Strep-tag sequence for accurate 37


molecular weight determinations on immunoblots.
Store at –20°C. 25
20
Catalog #: 161-0363
15

10
* Allow standards to reach room temperature, and mix thoroughly Stained with
to dissolve any precipitated solids. For certain applications, Coomassie
dilution of the standards is recommended. See instruction R-250.
manual for details.

5
Precision Plus Protein™ Standards,
All Blue
Size Range Quantity Recommended Load Volume

10 proteins, 10 –250 kD 750 µg protein Mini gels: 10 µl


in 500 µl of 30% (v/v) Large gels: 20 µl
glycerol, 2% SDS,
62.5 mM Tris, pH 6.8,
50 mM DTT, 5 mM EDTA,
0.02% NaN3

For electrophoretic transfer monitoring, and 250 kD


for improved molecular weight estimation on
SDS-polyacrylamide gels. 150

Recommended gel: 4 –20% gradient gel. See 100


protein migration chart on page 3 for approximate
75
migration distances with various gel percentages.

Note: Standards are premixed with sample loading 50

buffer. No dilution or heating is required.* 37


Prestained blue bands migrate to their true
molecular weight with no variability from lot to lot. 25

Store at –20°C. 20

15
Catalog #: 161-0373
10
* Allow standards to reach room temperature, and mix thoroughly
to dissolve any precipitated solids. For certain applications,
dilution of the standards is recommended. See instruction
manual for details.

6
Precision Plus Protein™ Standards,
Dual Color
Size Range Quantity Recommended Load Volume

10 proteins, 10 –250 kD 750 µg protein Mini gels: 10 µl


in 500 µl of 30% (v/v) Large gels: 20 µl
glycerol, 2% SDS,
62.5 mM Tris, pH 6.8,
50 mM DTT, 5 mM EDTA,
0.02% NaN3

For electrophoretic transfer monitoring, and 250 kD


for improved molecular weight estimation on
SDS-polyacrylamide gels. 150

Recommended gel: 4–20% gradient gel. See 100


protein migration chart on page 3 for approximate
75
migration distances with various gel percentages.
50
Note: Standards are premixed with sample loading
buffer. No dilution or heating is required.* Prestained 37
bands migrate to their true molecular weight with
no variability from lot to lot. Store at –20°C. 25
20
Catalog #: 161-0374 15

10

* Allow standards to reach room temperature, and mix thoroughly


to dissolve any precipitated solids. For certain applications,
dilution of the standards is recommended. See instruction
manual for details.

7
Unstained Standards
Protein Migration Chart

Representative migration of unstained standards on Ready Gel® Tris-HCl


pH 8.8 gels. Select the acrylamide percentage that best resolves your protein
or peptide of interest.

5% 7.5% 10% 12% 15% 18% 4–15% 4–20% 8–16% 10–20%

200 200
200 116 200
116 97.4 116
200 97.4 66
200 116 97.4
66 45 116
200 97.4 200 97.4
200 66
116
66 31 66
97.4 45
116 45
200 116 97.4 45
66
45 116
31 66
97.4 21.5 97.4 31
31
66 45

Standards
Unstained
66 45 31 21.5 14.4

31 21.5 21.5
116 45

14.4 21.5 14.4 14.4


6.5
97.4 31 21.5 31 14.4 6.5
45 6.5 6.5 6.5
21.5
66 14.4 14.4
6.5

Tris-HCI Gels

Protein molecular mass, kD. Migrations based on running gels until the dye front reached
the bottom of the gel.

8
SDS-PAGE Standards,
Broad Range
Size Range Quantity Recommended Load Volume

9 proteins, 6.5 –200 kD ~2.4 mg protein in Mini gels: 5 µl of


200 µl of 50% (v/v) a 1:20 dilution
glycerol, 300 mM NaCl, (Coomassie R-250 stain)
10 mM Tris, pH 8.5,
2 mM EDTA, 3 mM NaN3

For accurate molecular weight determination


Myosin 200 kD
on SDS-polyacrylamide gels. Blended to
give uniform band intensities when stained
-Galactosidase 116.3
with Coomassie R-250 or zinc stain. Phosphorylase b 97.4

Recommended gel: 4 –20% gradient gel. Bovine serum 66.2


albumin
See migration chart on page 8 for
approximate migration distances with Ovalbumin 45
various gel percentages.
Carbonic 31
Note: Dilute 1:20 in SDS-containing anhydrase
reducing sample buffer. Heat for 5 min at Soybean trypsin 21.5
inhibitor
95°C. Cool sample and load 10 µl/well for 14.4
Lysozyme
full-length gels or 5 µl/well for mini gels. Aprotinin 6.5
If you are silver staining, we recommend Stained with
Coomassie
using silver stain SDS-PAGE standards R-250.
(see pages 13–14). Store at –20°C.

Catalog #: 161-0317

9
SDS-PAGE Standards,
Low Range
Size Range Quantity Recommended Load Volume

6 proteins, 14.4 –97.4 kD ~2.4 mg protein in Mini gels: 5 µl of


200 µl of 50% (v/v) a 1:20 dilution
glycerol, 300 mM NaCl, (Coomassie R-250 stain)
10 mM Tris, pH 8.5,
2 mM EDTA, 3 mM NaN3

For accurate molecular weight determination Phosphorylase b 97.4 kD


on SDS-polyacrylamide gels. Blended to give
Bovine serum 66.2
uniform band intensities when stained with albumin
Coomassie R-250 or zinc stain.
Ovalbumin 45
Recommended gel: 12% acrylamide, or
gradient acrylamide gel with an upper
concentration of ≥12%. See migration chart
on page 8 for approximate migration distances Carbonic 31
with various gel percentages. anhydrase

Note: Dilute 1:20 in SDS-containing reducing


sample buffer. Heat for 5 min at 95°C. Cool
Soybean trypsin 21.5
sample and load 10 µl/well for full-length gels inhibitor
or 5 µl/well for mini gels. If you are silver
staining, we recommend using silver stain
SDS-PAGE standards (see pages 13–14). Lysozyme 14.4

Store at –20°C. Stained with


Coomassie
R-250.
Catalog #: 161-0304
10
SDS-PAGE Standards,
High Range
Size Range Quantity Recommended Load Volume

5 proteins, 45 –200 kD ~2.4 mg protein in Mini gels: 5 µl of


200 µl of 50% (v/v) a 1:20 dilution
glycerol, 300 mM NaCl, (Coomassie R-250 stain)
10 mM Tris, pH 8.5,
2 mM EDTA, 3 mM NaN3

For accurate molecular weight determination


on SDS-polyacrylamide gels. Blended to Myosin 200 kD
give uniform band intensities when stained
with Coomassie R-250 or zinc stain.

Recommended gel: 7.5% acrylamide. -Galactosidase 116.3


See migration chart on page 8 for
Phosphorylase b 97.4
approximate migration distances with
various gel percentages.

Note: Dilute 1:20 in SDS-containing Bovine serum 66.2


albumin
reducing sample buffer. Heat for 5 min at
95°C. Cool sample and load 10 µl/well for
full-length gels or 5 µl/well for mini gels. If
you are silver staining, we recommend
using silver stain SDS-PAGE standards
Ovalbumin 45
(see pages 13 –14). Store at –20°C.
Stained with
Coomassie
Catalog #: 161-0303 R-250.

11
Polypeptide SDS-PAGE Standards
Size Range Quantity Recommended Load Volume

6 proteins, 1.4 –26.6 kD ~5.4 mg protein in Mini gels: 5 µl of


200 µl of 40% (v/v) a 1:20 dilution
glycerol, 100 mM (Coomassie G-250 stain)
Tris-HCl, pH 8.5, 4 mM Large gels: 10 µl of
EDTA, 3 mM NaN3 a 1:20 dilution

For accurate molecular weight determination


of polypeptides and small proteins on
SDS-polyacrylamide gels. Blended to give Triosephosphate 26.6 kD
isomerase
uniform band intensities when stained with
Coomassie G-250 stain. Myoglobin 17.0

-Lactalbumin
Recommended gel: 16.5% Tris-Tricine or 14.4

10 –20% Tris-Tricine gradient gel. See


Aprotinin 6.5
migration chart on page 8 for approximate
migration distances with various gel Insulin chain B,
oxidized 3.5
percentages. Bacitracin 1.4

Note: Dilute 1:20 in Tris-Tricine sample Stained with


Coomassie
buffer. Heat for 5 min at 95°C. Cool G-250.
sample and load 10 µl/well for full-length
gels or 5 µl/well for mini gels. Store at
–20°C.

Catalog #: 161-0326

12
Silver Stain SDS-PAGE Standards,
Low Range
Size Range Quantity Recommended Load Volume

6 proteins, ~0.90 mg protein in Mini gels: 5 µl of


14.4 –97.4 kD 200 µl of 50% (v/v) a 1:20 dilution
glycerol, 20 mM Tris-HCl,
pH 8.5, 4 mM EDTA,
3 mM NaN3

Blended for even staining with silver


and other highly sensitive stains on
SDS-polyacrylamide gels.
Phosphorylase b 97.4 kD

Recommended gel: 12% acrylamide, or Bovine serum 66.2


albumin
gradient acrylamide gel with an upper
Ovalbumin 45
concentration of ≥12%. See migration chart
on page 8 for approximate migration
distances with various gel percentages.
Carbonic 31
anhydrase
Note: Dilute 1:20 in SDS-containing
reducing sample buffer. Heat for 5 min at
95°C. Cool sample and load 10 µl/well for Soybean trypsin 21.5
inhibitor
full-length gels or 5 µl/well for mini gels.
Store at –20°C.
Lysozyme 14.4
Catalog #: 161-0314
Stained
with Bio-Rad
silver stain.

13
Silver Stain SDS-PAGE Standards,
High Range
Size Range Quantity Recommended Load Volume

5 proteins, ~0.90 mg protein in Mini gels: 5 µl of


45 –200 kD 200 µl of 50% (v/v) a 1:20 dilution
glycerol, 20 mM Tris-HCl,
pH 8.5, 4 mM EDTA,
3 mM NaN3

Blended for even staining with silver


Myosin 200 kD
and other highly sensitive stains on
SDS-polyacrylamide gels.

Recommended gel: 7.5% acrylamide.


See migration chart on page 8 for -Galactosidase 116.3
approximate migration distances with
various gel percentages. Phosphorylase b 97.4

Note: Dilute 1:20 in SDS-containing


reducing sample buffer. Heat for 5 min at
Bovine serum 66.2
95°C. Cool sample and load 10 µl/well for albumin
full-length gels or 5 µl/well for mini gels.
Store at –20°C.

Catalog #: 161-0315
Ovalbumin 45

Stained
with Bio-Rad
silver stain.

14
SDS-PAGE Standards for
SYPRO Orange, Broad Range
Size Range Quantity Recommended Load Volume

9 proteins, ~1.8 mg protein in Mini gels: 5 µl of


6.5 –200 kD 200 µl of 50% (v/v) a 1:20 dilution
glycerol, 300 mM NaCl,
10 mM Tris, pH 8.5,
2 mM EDTA, 3 mM NaN3

For accurate molecular weight determination Myosin 200 kD


or as a staining reference on SYPRO Orange-
stained SDS-polyacrylamide gels. Protein
-Galactosidase 116.3
concentrations blended for uniform band
Phosphorylase b 97.4
staining with SYPRO Orange stain.
Bovine serum 66.2
Recommended gel: 4 –20% gradient gel. albumin
See migration chart on page 8 for
approximate migration distances with Ovalbumin 45

various gel percentages.

Note: Dilute 1:20 in SDS-containing Carbonic 31


anhydrase
reducing sample buffer. Heat for 5 min at
95°C. Cool sample and load 10 µl/well for Soybean trypsin 21.5
inhibitor
full-length gels or 5 µl/well for mini gels.
Lysozyme 14.4
Store at –20°C.
Aprotinin 6.5
Catalog #: 161-0332
Stained with
SYPRO Orange
fluorescent
protein stain.

15
Prestained Standards
Protein Migration Chart

Representative migration of prestained standards on Ready Gel® Tris-HCl pH 8.8


gels and Ready Gel Tris-Tricine gels. Select the acrylamide percentage that best
resolves your protein or peptide of interest.

5% 7.5% 10% 12% 15% 18% 4–15% 4–20% 8–16% 10–20% 8–16% 10–20%

200

Prestained
200

Standards
200 116 200
116 97.4 116
200 97.4 66
200 116 97.4
66 45 116 26.6
200 97.4 200 97.4
200 66
116
66 31 66 16.9
97.4 45 26.6
116 45 14.4
200 116 97.4 45
66
45 116 16.9
31 66
97.4 21.5 97.4 31
31
66 45
14.4
66 45 31 21.5 14.4

31 21.5 21.5
116 45
6.5
14.4 21.5 14.4 14.4
6.5
97.4 31 21.5 31 14.4 6.5
6.5
45 6.5 6.5 6.5
21.5 3.4
3.4
66 14.4 14.4 1.4 1.4
6.5

Tris-HCI Gels Tris-Tricine Gels

Protein molecular mass, kD.

16
Prestained SDS-PAGE Standards,
Broad Range
Size Range Quantity Recommended Load Volume

8 proteins, ~7.1–209 kD; ~0.63 mg protein in Mini gels: 10 µl


lot-specific molecular 500 µl of 33% (v/v) Mini blots: 5 µl
weights are included glycerol, 3% SDS, Large gels: 20 µl
with every vial 10 mM Tris, pH 7.0, Large blots: 10 µl
10 mM DTT, 2 mM
EDTA, 0.01% NaN3

For electrophoretic transfer monitoring and Myosin 210 kD


to estimate approximate molecular weights.

Recommended gel: 4 –20% gradient gel.


-Galactosidase 125
See migration chart on page 16 for
Bovine serum 101
approximate migration distances with albumin
various gel percentages.

Note: Standards are premixed with sample Ovalbumin 56.2

buffer. No heating or dilution is required.


Allow tube to reach room temperature, and Carbonic 35.8
anhydrase
mix thoroughly to dissolve any precipitated Soybean trypsin 29
inhibitor
solids. Store at –20°C.
Lysozyme 21
Catalog #: 161-0318
Aprotinin 6.9

Molecular weights
are representative of
this particular lot.

17
Prestained SDS-PAGE Standards,
Low Range
Size Range Quantity Recommended Load Volume

6 proteins, ~20 – 103 kD; ~0.63 mg protein in Mini gels: 10 µl


lot-specific molecular 500 µl of 33% (v/v) Mini blots: 5 µl
weights are included glycerol, 3% SDS, Large gels: 20 µl
with every vial 10 mM Tris, pH 7.0, Large blots: 10 µl
10 mM DTT, 2 mM
EDTA, 0.01% NaN3

For electrophoretic transfer monitoring and


Phosphorylase b 113 kD
to estimate approximate molecular weights.
Bovine serum 92
Recommended gel: 12% acrylamide. albumin

See migration chart on page 16 for


Ovalbumin 52.9
approximate migration distances with
various gel percentages.

Note: Standards are premixed with sample Carbonic anhydrase 35.4


buffer. No heating or dilution is required.
Allow tube to reach room temperature, and Soybean trypsin 29
mix thoroughly to dissolve any precipitated inhibitor

solids. Store at –20°C.

Catalog #: 161-0305 Lysozyme 21.5

Molecular weights
are representative
of this particular lot.

18
Prestained SDS-PAGE Standards,
High Range
Size Range Quantity Recommended Load Volume

4 proteins,~48 –204 kD; ~0.63 mg protein in Mini gels: 10 µl


lot-specific molecular 500 µl of 33% (v/v) Mini blots: 5 µl
weights are included glycerol, 3% SDS, Large gels: 20 µl
with every vial 10 mM Tris, pH 7.0, Large blots: 10 µl
10 mM DTT, 2 mM
EDTA, 0.01% NaN3

For electrophoretic transfer monitoring and


to estimate approximate molecular weights. Myosin 208 kD

Recommended gel: 7.5% acrylamide.


See migration chart on page 16 for 126
-Galactosidase
approximate migration distances with
various gel percentages. Bovine serum 97
albumin
Note: Standards are premixed with sample
buffer. No heating or dilution is required.
Allow tube to reach room temperature, and
mix thoroughly to dissolve any precipitated Ovalbumin 48
solids. Store at –20°C. Molecular weights
are representative
Catalog #: 161-0309 of this particular lot.

19
Kaleidoscope™ Prestained Standards
Size Range Quantity Recommended Load Volume

7 proteins, ~7–216 kD; ~1.6 mg protein in Mini gels: 10 µl


lot-specific molecular 500 µl of 33% (v/v) Mini blots: 5 µl
weights are included glycerol, 3% SDS, Large gels: 20 µl
with every vial 10 mM Tris, pH 7.0, Large blots: 10 µl
10 mM DTT, 2 mM
EDTA, 0.01% NaN3

Multicolored proteins for instant


Myosin 216 kD
band recognition on membranes or
SDS-polyacrylamide gels. -Galactosidase 132

Recommended gel: 4 –20% gradient gel.


Bovine serum 78
See migration chart on page 16 for albumin
approximate migration distances with
various gel percentages. Carbonic
45.7
anhydrase
Note: Standards are premixed with sample
buffer. No reconstitution or dilution is Soybean trypsin 32.5
inhibitor
required. Allow tube to reach room
temperature, and mix thoroughly to dissolve
any precipitated solids. Not intended for Lysozyme 18.4

precise molecular weight determinations. Aprotinin 7.6

Store at –20°C. Molecular weights


are representative
of this particular lot.
Catalog #: 161-0324

20
Kaleidoscope™ Polypeptide
Standards
Size Range Quantity Recommended Load Volume

5 proteins,~3.8 –36.4 kD; ~1.6 mg protein in Mini gels: 10 µl


lot-specific molecular 500 µl of 33% (v/v) Mini blots: 5 µl
weights are included glycerol, 0.5% SDS, Large gels: 20 µl
with every vial 10 mM Tris, pH 7.0, Large blots: 10 µl
10 mM DTT, 2 mM
EDTA, 0.01% NaN3

Multicolored proteins for instant band


recognition on membranes or Tricine Carbonic 36.4 kD
SDS-polyacrylamide gels. anhydrase
Soybean trypsin 26.6
inhibitor
Recommended gel: 15% Tris-Tricine.
See migration chart on page 16 for
Lysozyme 16
approximate migration distances with
varying gel percentages.

Note: Standards are premixed with sample Aprotinin 8.4


buffer. No reconstitution or dilution is
required. Allow tube to reach room
temperature, and mix thoroughly to dissolve Insulin chain B 3.8

any precipitated solids. Not intended for Molecular weights


are representative
precise molecular weight determinations. of this particular lot.
Store at –20°C.

Catalog #: 161-0325

21
Blotting Standards
Protein Migration Chart

Representative migration of blotting reference standards on Ready Gel® Tris-HCl

Standards
Blotting
pH 8.8 gels. Select the acrylamide percentage that best resolves your protein or
peptide of interest.

5% 7.5% 10% 12% 15% 18% 4–15% 4–20% 8–16% 10–20%

200 200
200 116 200
116 97.4 116
200 97.4 66
200 116 97.4
66 45 116
200 97.4 200 97.4
200 66
116
66 31 66
97.4 45
116 45
200 116 97.4 45
66
45 116
31 66
97.4 21.5 97.4 31
31
66 45

66 45 31 21.5 14.4

31 21.5 21.5
116 45

14.4 21.5 14.4 14.4


6.5
97.4 31 21.5 31 14.4 6.5
45 6.5 6.5 6.5
21.5
66 14.4 14.4
6.5

Tris-HCI Gels

Protein molecular mass, kD.

22
Biotinylated SDS-PAGE Standards,
Broad Range
Size Range Quantity Recommended Load Volume

9 proteins, ~0.13 mg protein in Mini gels: 10 µl of


6.5 –200 kD 250 µl of 50% (v/v) a 1:20 dilution
glycerol, 150 mM NaCl,
3 mM NaN3

For accurate molecular weight determination


Myosin 200 kD
of immunodetected proteins. The proteins
have been blended to give equal intensities
when detected with avidin-HRP or -AP -Galactosidase 116.3
Phosphorylase b 97.4
color development reagents.
Bovine serum 66.2
Recommended gel: 4 –20% gradient gel. albumin

See migration chart on page 22 for


approximate migration distances with Ovalbumin 45

various gel percentages.


Carbonic 31
Note: Dilute 1:4 (for HRP color development) anhydrase

or 1:20 (for AP color development) in Soybean trypsin 21.5


inhibitor
SDS-containing reducing sample buffer. Lysozyme 14.4
Heat for 5 min at 95°C. Cool sample and Aprotinin 6.5

load 10 –15 µl/well for full-length gels or Biotinylated


standards
10 µl/well for mini gels. Store at –20°C. transferred to
nitrocellulose
and detected
Catalog #: 161-0319 with avidin-AP.

23
Biotinylated SDS-PAGE Standards,
Low Range
Size Range Quantity Recommended Load Volume

6 proteins, ~0.13 mg protein in Mini gels: 2.5–10 µl


14.4 – 97.4 kD 250 µl of 50% (v/v) depending on method
glycerol, 150 mM NaCl, of detection
3 mM NaN3

For accurate molecular weight determination


of immunodetected proteins. The proteins
Phosphorylase b 97.4 kD
have been blended to give equal intensities Bovine serum 66.2
when detected with avidin-HRP or -AP albumin
Ovalbumin 45
color development reagents.

Recommended gel: 12% acrylamide.


Carbonic
See migration chart on page 22 for anhydrase
31

approximate migration distances with


various gel percentages. Soybean trypsin 21.5
inhibitor
Note: Dilute 1:4 (for HRP color development)
or 1:20 (for AP color development) in Lysozyme 14.4
SDS-containing reducing sample buffer.
Heat for 5 min at 95°C. Cool sample and
load 10–15 µl/well for full-length gels or
10 µl/well for mini gels. Store at –20°C.

Catalog #: 161-0306

24
Biotinylated SDS-PAGE Standards,
High Range
Size Range Quantity Recommended Load Volume

5 proteins, 45 –200 kD ~0.13 mg protein in Mini gels: 10 µl of


250 µl of 50% (v/v) a 1:20 dilution
glycerol, 150 mM NaCl,
3 mM NaN3

For accurate molecular weight determination


of immunodetected proteins. The proteins
Myosin 200 kD
have been blended to give equal intensities
when detected with avidin-HRP or -AP
color development reagent.
-Galactosidase 116.3
Recommended gel: 7.5% acrylamide.
Phosphorylase b 97.4
See migration chart on page 22 for
approximate migration distances with
Bovine serum 66.2
various gel percentages. albumin

Note: Dilute 1:4 (for HRP color development)


or 1:20 (forAP color development) in
SDS-containing reducing sample buffer.
Ovalbumin 45
Heat for 5 min at 95°C. Cool sample and
load 10 –15 µl/well for full-length gels or
10 µl/well for mini gels. Store at –20°C.

Catalog #: 161-0311

25
2-D and IEF
Standards
2-D and IEF Standards
Protein Migration Chart

Representative migration of IEF standards on wide and narrow pH range


Ready Gel® 5% polyacrylamide gels.

pH 3–10 pH 5–8

8.0 8.0
7.8
7.8

7.5
7.1
7.5
7.0
7.0

6.5

6.5

6.0 6.0

5.1
5.1

4.6

4.6

IEF Gels

Bands show pI values and migration representative of nondenaturing conditions.

26
2-D SDS-PAGE Standards
Range Quantity Recommended Load Volume

7 spots, 17.5–76.0 kD, ~0.30 mg protein in Mini gels: 2.5 µl


with pI 4.5 – 8.5 500 µl of 9 M urea, (Coomassie R-250 stain);
5% 2-mercaptoethanol, 0.5–2.5 µl (silver stain)
2% Bio-Lyte® 5/7 ampholyte

Use to determine pI and molecular weight pI


4.5 5.1 5.5 5.9 6.6 7.0 8.5
of sample proteins, to serve as a marker for
2-D gel matching, or as an internal control
76.0
to assess reproducibility. 66.2 1
Molecular mass, kD 2

Recommended gels: IEF slab or 43.0


3
36.0
ReadyStrip™ IPG strips for first dimension, 4
31.0
and PROTEAN® II precast gels for second 5
dimension. 21.5
6
17.5
Note: No dilution is required prior to use. 7

Visualize spots with silver stain, SYPRO Protein spots


Ruby protein gel stain, or Coomassie stain. 1. Conalbumin 5. Carbonic anhydrase
2. Albumin 6. Trypsin inhibitor
Store at –20°C. 3. Actin 7. Myoglobin
4. GAPDH
Catalog #: 161-0320

27
IEF Standards
Size Range Quantity Recommended Load Volume

9 proteins, 16 mg protein in Mini gels: 3 µl


pI 4.45 –9.6 250 µl of 50% (v/v) (Coomassie R-250
glycerol with 0.02% stain/Crocein Scarlet);
NaN3 0.5 µl (silver stain)

For pI calibration in analytical Lentil lectin


(3 bands)
polyacrylamide or agarose IEF gels. Five of
the nine proteins are naturally colored, pI 7.8, 8.0, 8.2
allowing continuous monitoring of the
Human
focusing process. pI 7.5 hemoglobin C
Human
pI 7.1
Recommended gel: Analytical pI 6.8, 7.0
hemoglobin A
Equine myoglobin
polyacrylamide or agarose IEF slab gel. (2 bands)
Human carbonic
Note: No reconstitution or dilution is pI 6.5 anhydrase
required prior to use. Not recommended
for 2-D electrophoretic applications.
Store at –20°C.
pI 6.0 Bovine carbonic
Catalog #: 161-0310 anhydrase
pI 5.1 -Lactoglobulin B

pI 4.45, 4.65, 4.75 Phycocyanin


(3 bands)

IEF standards run on Criterion™ IEF gel stained


with Bio-Rad IEF gel staining solution.
Cytochrome c, pI 9.6, not visible on this gel.

28
Protein Gel Stains
Gel Stain Sensitivity* Time Advantages

Coomassie R-250 36 – 47 ng 2.5 hr Simple, fast, consistent


Bio-Safe ™
8 –28 ng 2.5 hr Nonhazardous, water-only
Coomassie destain, eliminates MeOH waste
Zinc Stain 6 –12 ng 15 min Simple, fast, reversible,
high-contrast negative stain
Copper Stain 6 –12 ng 10 min Non-fixing, reversible,
single-reagent negative stain
SYPRO Orange 4– 8 ng 45 min Fluorescent stain
SYPRO Ruby 1–10 ng 3 hr Simple, highly sensitive
fluorescent stain
Silver Stain/ 0.5 –1.2 ng 75–120 min Highly sensitive protein and
Silver Stain Plus™ nucleic acid detection
IEF Stain 40 – 50 ng 3 hr Combines Coomassie R-250
and Crocein Scarlet stains
* Sensitivity is defined as the amount of protein in the faintest band.
Bio-Rad supplies these stains in convenient, ready-to-use solutions.

29
Molecular Weight Determination
Molecular weights of proteins are determined by comparison of their mobilities with
those of several marker proteins (standards) of known molecular weight:

1. After a gel has been run, but before it has been stained, mark the position of
the tracking dye.

2. After staining, measure the migration distance of each protein (standards and
unknowns) from the top of the resolving gel.

3. Calculate the relative mobility (R f) of each protein.


Distance migrated by protein
Rf =
Distance migrated by dye

4. Plot the R f of each standard protein


against the log10 of its molecular
weight, as shown, to generate
log MW

a standard curve.

Rf

5. To estimate the molecular weight of an unknown protein, use the standard


curve to interpolate its log10 molecular weight from its R f. Take the antilog
to determine its molecular weight.

30
Coomassie is a trademark of Imperial Chemical Industries PLC. Strep-tag and StrepTactin are
trademarks of Institut für Bioanalytik GmbH. SYPRO is a trademark of Molecular Probes, Inc.

Bio-Rad
Laboratories, Inc.

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