Abstract

One-dimensional gel electrophoresis of proteins provides information about the molecular size, amount, and purity of a protein sample. Separated proteins can be recovered from polyacrylamide gels for subsequent characterization by a variety of secondary techniques, such as mass spectrometry to determine post-translational modifications and the amino acid sequence. In addition, one-dimensional electrophoresis is the standard first step in immunoblotting and immunodetection. Protein separations in vertical slab gels are performed in a variety of formats. Most recently, small format minigels are typical due to their ease of use, low relative cost, and ready commercial availability. Larger gels provide more separation area and thus better resolution for complex samples and continue to be in use for specialized analysis. Curr. Protoc. Mol. Biol. 97:10.2A.1-10.2A.44. © 2012 by John Wiley & Sons, Inc.

Keywords: electrophoresis; SDS-PAGE; polyacrylamide; PAGE; minigel; protein; molecular weight; precast gels

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Introduction
Electricity and Electrophoresis
Basic Protocol 1: Denaturing (SDS) Discontinuous Gel Electrophoresis: Laemmli Gel Method
Alternate Protocol 1: Electrophoresis in Tris-Tricine Buffer Systems
Alternate Protocol 2: Nonurea Peptide Separations with Tris Buffers
Alternate Protocol 3: Continuous SDS-PAGE
Alternate Protocol 4: Casting and Running Ultrathin Gels
Support Protocol 1: Casting Multiple Single-Concentration Gels
Alternate Protocol 5: Separation of Proteins on Gradient Gels
Support Protocol 2: Casting Multiple Gradient Gels
Basic Protocol 2: Electrophoresis in Single-Concentration Minigels
Support Protocol 3: Preparing Multiple Gradient Minigels
Support Protocol 4: Calculating Molecular Mass
Reagents and Solutions
Commentary
Literature Cited
Figures
Tables

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Materials

Basic Protocol 1: Denaturing (SDS) Discontinuous Gel Electrophoresis: Laemmli Gel Method

Materials

Separating and stacking gel solutions (Table 10.2A.1)
H₂O-saturated isobutyl alcohol
1× Tris·Cl/SDS, pH 8.8 (dilute 4× Tris·Cl/SDS, pH 8.8; Table 10.2A.1)
Protein sample, on ice
2× and 1× SDS sample buffer (see recipe)
Protein molecular weight standards (Tables 10.2A.2 and 10.2A.3)
6× SDS sample buffer (see recipe; optional)
1× SDS electrophoresis buffer (see recipe)

Electrophoresis apparatus: e.g., Protean II 16-cm cell (Bio-Rad) or SE 600/400 16-cm unit (Hoefer) with clamps, glass plates, casting stand, and buffer chambers
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
Screw-top microcentrifuge tubes (recommended)
25- or 100-µl syringe with flat-tipped needle
Constant-current power supply (see Electricity and Electrophoresis above)

Alternate Protocol 1: Electrophoresis in Tris-Tricine Buffer Systems

Additional Materials (also see Basic Protocol 1)

Separating and stacking gel solutions (Table 10.2A.5)
2× tricine sample buffer (see recipe)
Peptide molecular weight standards (Table 10.2A.6)
Cathode buffer (see recipe)
Anode buffer (see recipe)
Coomassie blue G-250 staining solution (see recipe)
10% (v/v) acetic acid

50-ml Erlenmeyer side-arm flasks

Table 10.2A.5 Recipes for Tricine Peptide Separating and Stacking Gels^a

SEPARATING AND STACKING GELS

Stock solution^b	Separating gel	Stacking gel

30% acrylamide/0.8% bisacrylamide	9.80 ml	1.62 ml
Tris·Cl/SDS, pH 8.45	10.00 ml	3.10 ml
H₂O	7.03 ml	7.78 ml
Glycerol	4.00 g (3.17 ml)	—
10% (w/v) ammonium persulfate^c	50 µl	25 µl
TEMED	10 µl	5 µl

Prepare separating and stacking gel solutions separately.
In a 50-ml side-arm flask, mix 30% acrylamide/0.8% bisacrylamide solution (Table 10.2A.1), Tris·Cl/SDS, pH 8.45 (see reagents, below), and H₂O. Add glycerol to separating gel only. Degas under vacuum 10 to 15 min. Add 10% ammonium persulfate and TEMED. Swirl gently to mix; use immediately.
ADDITIONAL REAGENTS USED IN GELS
Tris·Cl/SDS, pH 8.45 (3.0 M Tris·Cl containing 0.3% SDS)
Dissolve 182 g Tris base in 300 ml H₂O. Adjust to pH 8.45 with 1 N HCl. Add H₂O to 500 ml total volume. Filter the solution through a 0.45-µm filter, add 1.5 g SDS, and store at 4°C up to 1 month.

^aThe recipes produce 30 ml of separating gel and 12.5 ml of stacking gel, which are adequate for two gels of dimensions 0.75 mm × 14 cm × 14 cm. The recipes are based on the Tris-tricine buffer system of Schagger and von Jagow (1987).

^bAll reagents and solutions used in the protocol must be prepared with Milli-Q-purified water or equivalent.

^cBest to prepare fresh. Failure to form a firm gel usually indicates a problem with the persulfate, TEMED, or both.

Table 10.2A.6 Molecular Weights of Peptide Standards for Polyacrylamide Gel Electrophoresis^a

Peptide	Molecular weight (Da)

Myoglobin (polypeptide backbone)	16,950
Myoglobin 1-131	14,440
Myoglobin 56-153	10,600
Myoglobin 56-131	8,160
Myoglobin 1-55	6,210
Glucagon	3,480
Myoglobin 132-153	2,510

^aPeptide standards are commercially available from Sigma-Aldrich. See Sigma-Aldrich Technical Bulletin MWSDS70-L for molecular weight markers for proteins.

Alternate Protocol 2: Nonurea Peptide Separations with Tris Buffers

Additional Materials (also see Basic Protocol 1)

Separating and stacking gel solutions (Table 10.2A.7)
2× Tris·Cl/SDS, pH 8.8 (dilute 4× Tris·Cl/SDS, pH 8.8; Table 10.2A.1)

2× SDS electrophoresis buffer (see recipe)

Table 10.2A.7 Recipes for Modified Laemmli Peptide Separating and Stacking Gels^a

SEPARATING AND STACKING GELS

Stock solution^b	Separating gel	Stacking gel

30% acrylamide/0.8% bisacrylamide	10.00 ml	0.65 ml
8× Tris·Cl, pH 8.8	3.75 ml	—
4× Tris·Cl, pH 6.8	—	1.25 ml
10% (w/v) SDS^c	0.15 ml	50 µl
H₂O	1.00 ml	3.00 ml
10% (w/v) ammonium persulfate^c	50 µl	25 µl
TEMED	10 µl	5 µl

Prepare separating and stacking gel solutions separately.
In a 25-ml side-arm flask, mix 30% acrylamide/0.8% bisacrylamide solution (see Table 10.2A.1), 8× Tris·Cl, pH 8.8, or 4× Tris·Cl, pH 6.8 (see reagents below), 10% SDS, and H₂O. Degas under vacuum 10 to 15 min. Add 10% ammonium persulfate and TEMED. Swirl gently to mix. Use immediately.
ADDITIONAL REAGENTS USED IN GELS
4× Tris·Cl, pH 6.8 (0.5 M Tris·Cl)
Dissolve 6.05 g Tris base in 40 ml H₂O. Adjust to pH 6.8 with 1 N HCl. Add H₂O to 100 ml total volume. Filter the solution through a 0.45-µm filter and store up to 1 month at 4°C.
8× Tris·Cl, pH 8.8 (3.0 M Tris·Cl)
Dissolve 182 g Tris base in 300 ml H₂O. Adjust to pH 8.8 with 1 N HCl. Add H₂O to 500 ml total volume. Filter the solution through a 0.45-µm filter and store up to 1 month at 4°C.

^aThe recipes produce 15 ml of separating gel and 5 ml of stacking gel, which are adequate for one gel of dimensions 0.75 mm × 14 cm × 14 cm. The recipes are based on the modified Laemmli peptide separation system of Okajima et al. (1993).

^bAll reagents and solutions used in the protocol must be prepared with Milli-Q-purified water or equivalent.

^cBest to prepare fresh. Failure to form a firm gel usually indicates a problem with the ammonium persulfate, TEMED, or both.

Alternate Protocol 3: Continuous SDS-PAGE

Additional Materials (also see Basic Protocol 1)

Separating gel solution (Table 10.2A.8)
2× and 1× phosphate/SDS sample buffer (see recipe)

1× phosphate/SDS electrophoresis buffer (see recipe)

Table 10.2A.8 Recipes for Separating Gels for Continuous SDS-PAGE^a

SEPARATING GEL

	Final acrylamide concentration in the separating gel (%)^c

Stock solution^b	5	6	7	8	9	10	11	12	13	14	15

30% acrylamide/0.8% bisacrylamide	2.5	3.00	3.50	4.00	4.50	5.00	5.50	6.00	6.50	7.00	7.50
4× phosphate/SDS, pH 7.2	3.75	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	7.25	6.75	6.25	5.75	5.25	4.75	4.25	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	0.05
TEMED	0.01	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 (see Table 10.2A.1), 4× phosphate/SDS, pH 7.2, and H₂O. Degas under vacuum about 5 min. Add 10% ammonium persulfate and TEMED. Swirl gently to mix. Use immediately.
ADDITIONAL REAGENTS USED IN GELS
4× phosphate/SDS, pH 7.2 (0.4 M sodium phosphate/0.4% SDS)
Mix 46.8 g NaH₂PO₄·H₂O, 231.6 g Na₂HPO₄·7H₂O, and 12 g SDS in 3 liters H₂O. Store up to 3 months at 4°C.

^aThe recipes produce 15 ml of separating gel, which is adequate for one gel of dimensions 0.75 mm × 14 cm × 14 cm. The recipes are based on the original continuous phosphate buffer system of Weber et al. (1972).

^bAll reagents and solutions used in the protocol must be prepared with Milli-Q-purified water or equivalent.

^cVolumes are in milliliters. The desired percentage of acrylamide in the separating gel depends on the molecular size of the protein being separated. See Basic Protocol 1, annotation to step 3.

^dBest to prepare fresh. Failure to form a firm gel usually indicates a problem with the ammonium persulfate, TEMED, or both.

Alternate Protocol 4: Casting and Running Ultrathin Gels

Additional Materials (also see Basic Protocol 1)

95% (v/v) ethanol
Gel Bond (FMC BioProducts) cut to a size slightly smaller than the gel plate dimensions
Glue stick
Ink roller (available from art supply stores)
Combs and spacers (0.19 to 0.5 mm; sequencing gel spacers and combs can be cut to fit)

Support Protocol 1: Casting Multiple Single-Concentration Gels

Additional Materials (also see Basic Protocol 1)

Separating and stacking gels for single-concentration gels (Table 10.2A.9)
Multiple gel caster (Bio-Rad, Hoefer)
100-ml disposable syringe and flat-tipped needle
Extra plates and spacers
14 × 14–cm acrylic blocks or polycarbonate sheets
250- and 500-ml side-arm flasks (used in gel preparation)
Long razor blade or plastic wedge (Wonder Wedge, Hoefer)

Resealable plastic bags

Table 10.2A.9 Recipes for Multiple Single-Concentration Polyacrylamide Gels^a

SEPARATING GEL

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

	5	6	7	8	9	10	11	12	13	14	15

30% acrylamide/0.8% bisacrylamide	52	62	72	83	93	103	114	124	134	145	155
4× Tris·Cl/SDS, pH 8.8	78	78	78	78	78	78	78	78	78	78	78
H₂O	181	171	160	150	140	129	119	109	98	88	78
10% (w/v) ammonium persulfate^d	1.0	1.0	1.0	1.0	1.0	1.0	1.0	1.0	1.0	1.0	1.0
TEMED	0.21	0.21	0.21	0.21	0.21	0.21	0.21	0.21	0.21	0.21	0.21

Preparation of separating gel
In a 500-ml side-arm flask, mix 30% acrylamide/0.8% bisacrylamide solution (see Table 10.2A.1), 4× Tris·Cl/SDS, pH 8.8 (Table 10.2A.1), and H₂O. Degas under vacuum ~5 min. Add 10% ammonium persulfate and TEMED. Swirl gently to mix. Use immediately.
STACKING GEL
In a 250-ml side-arm flask, mix 13.0 ml 30% acrylamide/0.8% bisacrylamide solution, 25 ml 4× Tris·Cl/SDS, pH 6.8 (Table 10.2A.1), and 61 ml H₂O. Degas under vacuum ~5 min. Add 0.25 ml 10% ammonium persulfate and 50 µl TEMED. Swirl gently to mix. Use immediately.

^aThe recipes produce about 300 ml of separating gel and 100 ml of stacking gel, which are adequate for ten gels of dimensions 1.5 mm × 14 cm × 14 cm with extra solution should there be a leak or spill while casting the gels. For thinner spacers or fewer gels, calculate volumes using the equation in the annotation to step 4. The recipes are based on the SDS (denaturing) discontinuous buffer system of Laemmli (1970).

^bAll reagents and solutions used in the protocol must be prepared with Milli-Q-purified water or equivalent.

^cVolumes in table body are in milliliters. The desired percentage of acrylamide in separating gel depends on the molecular size of the protein being separated. See Basic Protocol 1, annotation to step 3.

^dBest to prepare fresh. Failure to form a firm gel usually indicates a problem with the persulfate, TEMED, or both.

Alternate Protocol 5: Separation of Proteins on Gradient Gels

Additional Materials (also see Basic Protocol 1)

Light and heavy acrylamide gel solutions (Table 10.2A.10)
Bromphenol blue (optional; for checking practice gradient)
10% ammonium persulfate (prepare fresh)
TEMED

Gradient maker (30 to 50 ml, Hoefer SG30 or SG50; or 30 to 100 ml, Bio-Rad 385)
Tygon tubing with micropipet tip
Peristaltic pump (optional; e.g., Markson A-13002, A-34040, or A-34105 minipump)

Whatman 3MM filter paper

Table 10.2A.10 Light and Heavy Acrylamide Gel Solutions for Gradient Gels

	Acrylamide concentration in light gel solution (%)^a,b

Stock solution	5	6	7	8	9	10	11	12	13	14

30% acrylamide/0.8% bisacrylamide^c	2.5	3.0	3.5	4.0	4.5	5.0	5.5	6.0	6.5	7.0
4× Tris·Cl/SDS, pH 8.8^c	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	7.25	6.75	6.25	5.75	5.25	4.75	4.25
10% ammonium persulfate^d	0.05	0.05	0.05	0.05	0.05	0.05	0.05	0.05	0.05	0.05
TEMED^d	0.005	0.005	0.005	0.005	0.005	0.005	0.005	0.005	0.005	0.005

	Acrylamide concentration in heavy gel solution (%)^a,b

Stock solution	10	11	12	13	14	15	16	17	18	19	20

30% acrylamide/0.8% bisacrylamide^c	5.0	5.5	6.0	6.5	7.0	7.5	8.0	8.5	9.0	9.5	10.0
4× Tris·Cl/SDS, pH 8.8^c	3.75	3.75	3.75	3.75	3.75	3.75	3.75	3.75	3.75	3.75	3.75
H₂O	5.0	4.5	4.0	3.5	3.0	2.5	2.0	1.5	1.0	0.5	0
Sucrose (g)	2.25	2.25	2.25	2.25	2.25	2.25	2.25	2.25	2.25	2.25	2.25
10% ammonium persulfate^d	0.05	0.05	0.05	0.05	0.05	0.05	0.05	0.05	0.05	0.05	0.05
TEMED^d	0.005	0.005	0.005	0.005	0.005	0.005	0.005	0.005	0.005	0.005	0.005

^aTo survey proteins ³10 kDa, 5% to 20% gradient gels are recommended. To expand the range between 10 and 200 kDa, a 10% to 20% gradient gel is recommended.

^bVolumes are in milliliters (sucrose is in grams). Keep light gel solution at room temperature prior to use (no longer than 1 hr). Keep heavy solution on ice.

^cSee Table 10.2A.1 for preparation.

^dAmmonium persulfate and TEMED are added directly to the gradient chambers immediately before the gel is poured. It is best to prepare ammonium persulfate fresh. Failure to form a firm gel usually indicates a problem with the ammonium persulfate, TEMED, or both.

Support Protocol 2: Casting Multiple Gradient Gels

Additional Materials (also see Alternate Protocol 5)

Plug solution (see recipe)
Light and heavy acrylamide gel solutions for multiple gradient gels (Table 10.2A.11)
TEMED
H₂O-saturated isobutyl alcohol

Multiple gel caster (Bio-Rad, Hoefer)
Peristaltic pump (25 ml/min)
500- or 1000-ml gradient maker (Bio-Rad, Hoefer)

Tygon tubing

Table 10.2A.11 Light and Heavy Acrylamide Gel Solutions for Casting Multiple Gradient Gels

	Acrylamide concentration in light gel solution (%)^a,b

Stock solution	5	6	7	8	9	10	11	12	13	14

30% acrylamide/0.8% bisacrylamide^c	28	33	39	44	50	55	61	66	72	77
4× Tris·Cl/SDS, pH 8.8^c	41	41	41	41	41	41	41	41	41	41
H₂O	96	91	85	80	74	69	63	58	52	47
10% ammonium persulfate^d	0.55	0.55	0.55	0.55	0.55	0.55	0.55	0.55	0.55	0.55
TEMED	0.054	0.054	0.054	0.054	0.054	0.054	0.054	0.054	0.054	0.054

	Acrylamide concentration in heavy gel solution (%)^a,b

Stock solution	10	11	12	13	14	15	16	17	18	19	20

30% acrylamide/0.8% bisacrylamide^c	55	61	66	72	77	83	88	94	99	105	110
4× Tris·Cl/SDS, pH 8.8^c	41	41	41	41	41	41	41	41	41	41	41
H₂O	55	50	44	39	33	28	22	17	11	5.5	0
Sucrose (g)	25	25	25	25	25	25	25	25	25	25	25
10% ammonium persulfate^d	0.55	0.55	0.55	0.55	0.55	0.55	0.55	0.55	0.55	0.55	0.55
TEMED	0.054	0.054	0.054	0.054	0.054	0.054	0.054	0.054	0.054	0.054	0.054

^aTo survey proteins ³10 kDa, 5% to 20% gradient gels are recommended. To expand the range between 10 and 200 kDa, a 10% to 20% gradient gel is recommended.

^bVolumes are in milliliters (sucrose is in grams). Recipes produce ten 1.5-mm-thick gradient gels with 10 ml extra solution to account for losses in tubing. Keep light gel solution at room temperature prior to use (no longer than 1 hr). Keep heavy solution on ice.

^cSee Table 10.2A.1 for preparation.

^dBest to prepare fresh. Failure to form a firm gel usually indicates a problem with the ammonium persulfate, TEMED, or both.

Basic Protocol 2: Electrophoresis in Single-Concentration Minigels

Materials

Minigel vertical gel unit (Hoefer Mighty Small SE 250/280 or Bio-Rad Mini-Protean II) with glass plates, clamps, and buffer chambers
0.75-mm spacers
Multiple gel caster (Hoefer SE-275/295 or Bio-Rad Mini-Protean II multicasting chamber)
Acrylic plate (Hoefer SE-217 or Bio-Rad 165-1957) or polycarbonate separation sheet (Hoefer SE-213 or Bio-Rad 165-1958)
10- and 50-ml syringes
Combs (Teflon, Hoefer SE-211A series or Bio-Rad Mini-Protean II)
Long razor blade
Micropipet

Additional reagents and equipment for standard denaturing SDS-PAGE (see Basic Protocol 1)

Support Protocol 3: Preparing Multiple Gradient Minigels

Additional Materials (also see Basic Protocol 2)

Plug solution (see recipe)

Additional reagents and equipment for preparing gradient gels (see Alternate Protocol 5)

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Figures

Figure 10.2A.1 Series and parallel connections of gel tanks to power supply.

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Figure 10.2A.2 Gradient gel setup. A peristaltic pump, though not required, will provide better control.

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Figure 10.2A.3 Setup for casting multiple gradient gels. Casting multiple gradient gels requires a peristaltic pump and a multiple gel caster. Gel solution is introduced through the bottom of the multiple caster.

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Figure 10.2A.4 Minigel sandwiches positioned in the multiple gel caster. Extra glass or acrylic plates or polycarbonate sheets are used to fill any free space in the caster and to ensure that the gel sandwiches are held firmly in place.

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Figure 10.2A.5 Example of an R_f calculator. This sheet is copied to transparency film using a paper copier and used as an overlay on the gel. When the transparency is placed on top of the gel, so that the top of the gel aligns with the top of the calculator and the dye front aligns with the bottom of the calculator, the R_f can be read directly off the overlay. Note that the calculator accommodates a range of gel lenxgths. The overlay should be copied at a 1:1 ratio so that the centimeter scale remains accurate. However, as long as the overlay can fit the top and bottom of the gel, the R_f numbers will be accurate.

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Figure 10.2A.6 Standard protein molecular weight curves for (A) single-concentration (5% and 12.5%) and (B) gradient (5% to 20%) gels. Protein standards are separated via SDS-PAGE, visualized by staining with Coomassie blue (unit 10.6), and measured relative to the dye front to give the relative mobility (R_f). Note the single-concentration gel has a more limited range of linearity than the gradient gel. The standard curve permits the calculation of the molecular weight of an unknown by using the R_f of the unknown to predict the molecular weight.

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Figure 10.2A.7 Structures of acrylamide and bisacrylamide and the associated reaction producing the polyacrylamide matrix used for protein separation.

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Figure 10.2A.8 Structures of sodium dodecyl sulfate, dithiothreitol, and 2-mercaptoethanol: used to break disulfide bonds in proteins so they are fully denatured.

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Figure 10.2A.9 Separation of membrane proteins by 5.1% to 20.5% T polyacrylamide gradient SDS-PAGE. Approximately 30 µl of 1× SDS sample buffer containing 30 µg of Alaskan pea (Pisum sativum) membrane proteins was loaded in wells of a 14 × 14–cm, 0.75-mm-thick gel. Standard proteins were included in the outside lanes. The gel was run at 4 mA for »15 hr.

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Figure 10.2A.10 A typical version of a form used for recording gel data.

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Literature Cited

Literature Cited
	Dhugga, K.S., Waines, J.G., and Leonard, R.T. 1988. Correlated induction of nitrate uptake and membrane polypeptides in corn roots. Plant Physiol. 87:120-125.
	Gallagher, S.R. and Leonard, R.T. 1987. Electrophoretic characterization of a detergent-treated plasma membrane fraction from corn roots. Plant Physiol. 83:265-271.
	Hunkapiller, M.W., Lujan, E., Ostrander, F., and Hood, L.E. 1983. Isolation of microgram quantities of proteins from polyacrylamide gels for amino acid sequence analysis. Methods Enzymol. 91:227-236.
	Laemmli, U.K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680-685.
	Matsudaira, P.T. and Burgess, D.R. 1978. SDS microslab linear gradient polyacrylamide gel electrophoresis. Anal. Biochem. 87:386-396.
	Okajima, T., Tanabe, T., and Yasuda, T. 1993. Nonurea sodium dodecyl sulfate-polyacrylamide gel electrophoresis with high-molarity buffers for the separation of proteins and peptides. Anal. Biochem. 211:293-300.
	Ploegh, H.L. 1995. One-Dimensional Isoelectric Focusing of Proteins in Slab Gels. In Current Protocols in Protein Science (J.E. Coligan, B.M. Dunn, D.W. Speicher, and P.T. Wingfield, eds.) pp. 10.2.1-10.2.8. John Wiley & Sons, Hoboken, N.J.
	Schagger, H. and von Jagow, G. 1987. Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa. Anal. Biochem. 166:368-379.
	See, Y.P., Olley, P.M., and Jackowski, G. 1985. The effects of high salt concentrations in the samples on molecular weight determination in sodium dodecyl sulfate polyacrylamide gel electrophoresis. Electrophoresis 8:382-387.
	Takano, E., Maki, M., Mori, H., Hatanaka, N., Marti, T., Titani, K., Kannagi, R., Ooi, T., and Murachi, T. 1988. Pig heart calpastatin: Identification of repetitive domain structures and anomalous behavior in polyacrylamide gel electrophoresis. Biochemistry 27:1964-1972.
	Weber, K., Pringle, J.R., and Osborn, M. 1972. Measurement of molecular weights by electrophoresis on SDS-acrylamide gel. Methods Enzymol. 26:3-27.
Key Reference
	Hames, B.D. and Rickwood, D. (eds.) 1990. Gel Electrophoresis of Proteins: A Practical Approach, 2nd ed. Oxford University Press, New York.
An excellent book describing gel electrophoresis of proteins.

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One‐Dimensional SDS Gel Electrophoresis of Proteins

Abstract

Table of Contents

Materials

Basic Protocol 1: Denaturing (SDS) Discontinuous Gel Electrophoresis: Laemmli Gel Method

Alternate Protocol 1: Electrophoresis in Tris-Tricine Buffer Systems

Alternate Protocol 2: Nonurea Peptide Separations with Tris Buffers

Alternate Protocol 3: Continuous SDS-PAGE

Alternate Protocol 4: Casting and Running Ultrathin Gels

Support Protocol 1: Casting Multiple Single-Concentration Gels

Alternate Protocol 5: Separation of Proteins on Gradient Gels

Support Protocol 2: Casting Multiple Gradient Gels

Basic Protocol 2: Electrophoresis in Single-Concentration Minigels

Support Protocol 3: Preparing Multiple Gradient Minigels

Figures

Literature Cited

Website Resources

Troubleshooting Tips

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One‐Dimensional SDS Gel Electrophoresis of Proteins

Abstract

Table of Contents

Materials

Basic Protocol 1: Denaturing (SDS) Discontinuous Gel Electrophoresis: Laemmli Gel Method

Alternate Protocol 1: Electrophoresis in Tris-Tricine Buffer Systems

Alternate Protocol 2: Nonurea Peptide Separations with Tris Buffers

Alternate Protocol 3: Continuous SDS-PAGE

Alternate Protocol 4: Casting and Running Ultrathin Gels

Support Protocol 1: Casting Multiple Single-Concentration Gels

Alternate Protocol 5: Separation of Proteins on Gradient Gels

Support Protocol 2: Casting Multiple Gradient Gels

Basic Protocol 2: Electrophoresis in Single-Concentration Minigels

Support Protocol 3: Preparing Multiple Gradient Minigels

Figures

Videos

Literature Cited

Website Resources

Troubleshooting Tips

Most Popular Tools