Protein Analysis by SDS‐PAGE and Detection by Coomassie Blue or Silver Staining

Sean Gallagher (electrophoresis, staining)¹, Joachim Sasse (staining)²

¹ Motorola Phoenix Applied Research Center, Tempe, Arizona, ² Shriners Hospital, Tampa, Florida

Publication Name: Current Protocols in Pharmacology

Unit Number: Appendix 3B

DOI: 10.1002/0471141755.pha03bs02

Online Posting Date: May, 2001

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Abstract

Electrophoresis is used to separate complex mixtures of proteins, to investigate subunit compositions and to verify homogeneity of protein samples. It can also serve to purify proteins for use in further applications. In polyacrylamide gel electrophoresis, the combination of gel pore size and protein charge, size, and shape determines the migration rate of the protein. This unit first presents an informative section about safe use of electricity and electrophoresis. Protocols are then provided for one-dimensional gel electrophoresis under denaturing conditions and subsequent staining with either Coomassie blue or silver staining.Electrophoresis is used to separate complex mixtures of proteins, to investigate subunit compositions and to verify homogeneit

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Unit Introduction
Electricity and Electrophoresis
Basic Protocol: Denaturing (SDS) Discontinuous Gel Electrophoresis: Laemmli Gel Method
Staining Proteins in Gels
Support Protocol 1: Coomassie Blue Staining
Support Protocol 2: Silver Staining
Reagents and Solutions
Figures
Tables

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Materials

Basic Protocol: Denaturing (SDS) Discontinuous Gel Electrophoresis: Laemmli Gel Method

Materials

Separating and stacking gel solutions (Table A.3B.1)
H₂O-saturated isobutyl alcohol
1× Tris×Cl/SDS, pH 8.8 (dilute 4× Tris×Cl/SDS, pH 8.8; see recipe)
Protein sample to be analyzed
2× and 1× SDS sample buffer (see recipe)
Protein-molecular-weight standards mixture (e.g., Bio-Rad; see Table A.3B.2)
6× SDS sample buffer (optional)

1× SDS electrophoresis buffer (see recipe)

Table A.3B.2 Molecular Weights of Protein Standards for Polyacrylamide Gel Electrophoresis^a

Protein	Molecular weight

Cytochrome c	11,700
-Lactalbumin	14,200
Lysozyme (hen egg white)	14,300
Myoglobin (sperm whale)	16,800
-Lactoglobulin	18,400
Trypsin inhibitor (soybean)	20,100
Trypsinogen, PMSF treated	24,000
Carbonic anhydrase (bovine erythrocytes)	29,000
Glyceraldehyde-3-phosphate dehydrogenase (rabbit muscle)	36,000
Lactate dehydrogenase (porcine heart)	36,000
Aldolase	40,000
Ovalbumin	45,000
Catalase	57,000
Bovine serum albumin	66,000
Phosphorylase b (rabbit muscle)	97,400
-Galactosidase	116,000
RNA polymerase, E. coli	160,000
Myosin, heavy chain (rabbit muscle)	205,000

^a Protein standards are commercially available in kits (e.g., Hoefer Pharmacia Biotech, Life Technologies, Bio-Rad, or Sigma).

Electrophoresis apparatus with clamps, glass plates, casting stand, and buffer chambers (Bio-Rad, Hoefer Pharmacia Biotech)
0.75-mm spacers
0.45-µm filters (used in stock solution preparation)
25-ml Erlenmeyer side-arm flasks
Vacuum pump with cold trap
0.75-mm Teflon comb with 1, 3, 5, 10, 15, or 20 teeth
1.5-ml screw-cap microcentrifuge tube
25- or 100-µl syringe with flat-tipped needle

Constant-current power supply

Table A.3B.1 Recipes for Polyacrylamide Separating and Stacking Gels^a

SEPARATING GEL

Stock solution^b	Final acrylamide concentration in separating gel (%)^c

	5	6	7	7.5	8	9	10	12	13	15

30% acrylamide/0.8% bisacrylamide	2.50	3.00	3.50	3.75	4.00	4.50	5.00	6.00	6.50	7.50
4× Tris×Cl/SDS pH 8.8	3.75	3.75	3.75	3.75	3.75	3.75	3.75	3.75	3.75	3.75
H₂O	8.75	8.25	7.75	7.50	7.25	6.75	6.25	5.25	4.75	3.75
10% (w/v) ammonium persulfate^d	0.05	0.05	0.05	0.05	0.05	0.05	0.05	0.05	0.05	0.05
TEMED	0.01	0.01	0.01	0.01	0.01	0.01	0.01	0.01	0.01	0.01

Preparation of separating gel
In a 25-ml side-arm flask, mix 30% acrylamide/0.8% bisacrylamide solution, 4× Tris×Cl/SDS, pH 6.8 (see recipe), and H₂O in the amounts specified above. Degas under vacuum about 5 min. Add the 10% ammonium persulfate solution and TEMED (N,N,N¢,N¢-tetramethylethylenediamine). Swirl gently to mix. Use immediately.
STACKING GEL (3.9% acrylamide)
In a 25-ml side-arm flask, mix 0.65 ml of 30% acrylamide/0.8% bisacrylamide, 1.25 ml of 4× Tris×Cl/SDS, pH 6.8 (see recipe), and 3.05 ml H₂O. Degas under vacuum 10 to 15 min. Add 25 µl of 10% ammonium persulfate and 5 µl TEMED. Swirl gently to mix. Use immediately. Failure to form a firm gel usually indicates a problem with the persulfate, TEMED, or both.

^a The recipes produce 15 ml of separating gel and 5 ml of stacking gel, which are adequate for a gel of dimensions 0.75 mm × 14 cm × 14 cm. The recipes are based on the SDS (denaturing) discontinuous buffer system of Laemmli (1970).

^b All reagents and solutions used in the protocol must be prepared with water purified using a Milli-Q-filter (Millipore) or equivalent. See Reagents and Solutions for recipes.

^c Units of numbers in table body are milliliters. The desired percentage of acrylamide in the separating gel depends on the molecular size of the protein being separated. See annotation to step .

^d Best to prepare fresh.

Support Protocol 1: Coomassie Blue Staining

Materials

Polyacrylamide gel (see Basic Protocol)
Fixing solution (see recipe)
Coomassie staining solution (see recipe)
Destaining solution (see recipe)
7% (v/v) aqueous acetic acid

Cellophane membrane backing sheets (optional; Hoefer Pharmacia Biotech or Bio-Rad)
Appropriate photographic filter and film (optional): yellow-orange filter (e.g., Wratten no. 8 or no. 9 filter series A) with fine-grained panchromatic halftone film (e.g., Kodak T-Max 100)
Whatman 3MM filter paper (optional)

NOTE: Deionized, distilled water should be used throughout this protocol.

Support Protocol 2: Silver Staining

Materials

Fixing and destaining solutions (see recipe)
10% (v/v) glutaraldehyde (freshly prepared from 50% stock; Eastman Kodak)
Silver nitrate solution (ammoniacal; see recipe)
Developing solution (see recipe)
Kodak Rapid Fix Solution A
Appropriate photographic filter and film (optional): blue-green filter (e.g., Wratten no. 58) with Kodak T-Max 100 or equivalent

NOTE: Wear gloves at all times to avoid fingerprint contamination.

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Figures

Figure A.3B.1 Series and parallel connections of gel tanks to power supply.

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Figure A.3B.2 Typical calibration curves obtained with standard proteins separated by nongradient denaturing (SDS) discontinuous gel electrophoresis based on the method of Laemmli. (A) Gel with 7% polyacrylamide. (B) Gel with 11% polyacrylamide. (Redrawn with permission from Sigma.)

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Literature Cited

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