One‐Dimensional SDS Gel Electrophoresis of Proteins

Sean R. Gallagher¹

¹ UVP, LLC, Upland, California

Publication Name: Current Protocols in Protein Science

Unit Number: Unit 10.1

DOI: 10.1002/0471140864.ps1001s68

Online Posting Date: April, 2012

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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. Protein Sci. 68:10.1.1‐10.1.44. © 2012 by John Wiley & Sons, Inc.

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

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Table of Contents

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 10.1.1)
H₂O‐saturated isobutyl alcohol
1× Tris·Cl/SDS, pH 8.8 (dilute 4× Tris·Cl/SDS, pH 8.8; Table 10.1.1)
Protein sample, on ice
2× and 1× SDS sample buffer (see recipe)
Protein molecular weight standards (Tables 10.1.2 and 10.1.3)
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 10.1.5)
2× tricine sample buffer (see recipe)
Peptide molecular weight standards (Table 10.1.6)
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

Alternate Protocol 2: Nonurea Peptide Separations with Tris Buffers

Additional Materials (also see Basic Protocol 1)

Separating and stacking gel solutions (Table 10.1.7)
2× Tris·Cl/SDS, pH 8.8 (dilute 4× Tris·Cl/SDS, pH 8.8; Table 10.1.1)
2× SDS electrophoresis buffer (see recipe)

Alternate Protocol 3: Continuous SDS‐PAGE

Additional Materials (also see Basic Protocol 1)

Separating gel solution (Table 10.1.8)
2× and 1× phosphate/SDS sample buffer (see recipe)
1× phosphate/SDS electrophoresis buffer (see recipe)

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 10.1.9)
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

Alternate Protocol 5: Separation of Proteins on Gradient Gels

Additional Materials (also see Basic Protocol 1)

Light and heavy acrylamide gel solutions (Table 10.1.10)
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

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 10.1.11)
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

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 10.1.1 Series and parallel connections of gel tanks to power supply.

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Figure 10.1.2 Gradient gel setup. A peristaltic pump, though not required, will provide better control.

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Figure 10.1.3 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.

Figure 10.1.4 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.

Figure 10.1.5 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.

Figure 10.1.6 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 10.5), 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.

Figure 10.1.7 Structures of acrylamide and bisacrylamide and the associated reaction producing the polyacrylamide matrix used for protein separation.

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Figure 10.1.8 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 10.1.9 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.

Figure 10.1.10 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 vonJagow, 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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