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SDS‐Polyacrylamide Gel Electrophoresis (SDS‐PAGE)
Publication Name:
Current Protocols Essential Laboratory Techniques
Unit Number:
Unit 7.3
DOI:
10.1002/9780470089941.et0703s06
Online Posting Date:
September, 2012
Abstract
Electrophoresis is used to separate complex mixtures of proteins (e.g., from cells, subcellular fractions, column fractions, or immunoprecipitates), investigate subunit compositions, verify homogeneity of protein samples, and purify proteins for use in further applications. In polyacrylamide gel electrophoresis, proteins migrate in response to an electrical field through pores in a polyacrylamide gel matrix; pore size decreases with increasing acrylamide concentration. The combination of pore size and protein charge, size, and shape determines the migration rate of the protein. In this unit, the standard Laemmli method is described for discontinuous gel electrophoresis under denaturing conditions, i.e., in the presence of sodium dodecyl sulfate (SDS). Support protocols cover the casting of gels, calculation of molecular mass using the electrophoretic mobility of a protein, and purification of SDS by recrystallization. Curr. Protoc. Essential Lab. Tech. 6:7.3.1-7.3.28. © 2012 by John Wiley & Sons, Inc.
Keywords: protein; electrophoresis; separation; polyacrylamide; SDS-PAGE
Table of Contents
- Overview and Principles
- Strategic Questions
- Strategic Planning
- Safety Considerations
- Protocols
- Basic Protocol: Denaturing (SDS) Discontinuous Gel Electrophoresis: The Laemmli Gel Method
- Support Protocol 1: Casting a Gel for Use in Denaturing Discontinuous Electrophoresis
- Support Protocol 2: Calculating Molecular Mass
- Support Protocol 3: Recrystallizing SDS
- Reagents and Solutions
- Understanding Results
- Troubleshooting
- Variations
- Literature Cited
- Figures
- Tables
Materials
Basic Protocol: Denaturing (SDS) Discontinuous Gel Electrophoresis: The Laemmli Gel Method Materials
CAUTION: The voltages and currents used during electrophoresis are dangerous and potentially lethal. It is extremely important to read the section entitled Safety Considerations before performing any electrophoresis. Support Protocol 1: Casting a Gel for Use in Denaturing Discontinuous Electrophoresis Materials
Support Protocol 2: Calculating Molecular Mass Materials
Support Protocol 3: Recrystallizing SDS Materials
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Figures
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Figure 7.3.1 (A) Structure of sodium dodecyl sulfate. (B) SDS coating a denatured protein chain illustrating the constant binding of SDS per unit length of protein. (C) Dithiothreitol and (D) 2-mercaptoethanol, reductants used to break disulfide bonds in proteins so they are fully denatured. -
Figure 7.3.2 Standard curves for (A) a gradient 5% to 20% gel and (B) two single-concentration gels. Protein standards were separated via SDS-PAGE and visualized by staining with the protein-specific stain Coomassie Blue (unit f ). Note the single-concentration gels have a more limited range of linearity for molecular-weight measurements than the gradient gel. The standard curves permit the calculation of the molecular weight of an unknown by simply using the Rf of the unknown to predict the molecular weight. -
Figure 7.3.3 Free radical chain polymerization of acrylamide and N,N¢-methylenebisacrylamide. Polymerization is initiated via free radicals generated by a redox pair, ammonium persulfate, and N,N,N¢,N¢-tetramethylethylenediamine (TEMED). Polymerizing the acrylamide monomer by itself produces long linear chains of polymer in the form of a thick liquid with a syrup-like consistency. By adding the N,N¢-methylenebisacrylamide, a cross-link with four potential branch points for the linear chain is copolymerized with the acrylamide to yield a mesh or gel. Note that the reaction is exothermic, and the outside of the gel plate will be warm during polymerization. The mesh creates pores of varying size ranges, depending on the concentration of acrylamide and N,N¢-methylenebisacrylamide, to produce the sieving gel that is used for protein separation. -
Figure 7.3.4 Computerized analysis of separated proteins. Acquiring the image of the stained protein gel with a digital camera greatly simplifies subsequent analysis. The protein bands are automatically identified, assigned an R f , and given an estimated molecular mass via comparison to the standard curve generated from the separated standard proteins. The gel analysis software (right panel) illustrated in this figure is Vision Works LS (UVP). SigmaPlot, a graphing package, is illustrated in the left panel. -
Figure 7.3.5 Example of a relative mobility (R f ) calculator. This sheet can be 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 lines up with the bottom of the calculator, the Rf can be read directly off the overlay. Note that the calculator accommodates a range of gel lengths. 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 Rf numbers will be accurate. -
Figure 7.3.6 Enriched plasma membrane fractions from corn were separated by denaturing gradient SDS-PAGE. Lanes 1 and 3 contain denatured and reduced proteins, while 2 and 4 are the same samples which, while denatured, have not been reduced. Note that reducing the proteins generated darker bands of lower molecular-weight proteins, indicating the presence of a larger multisubunit complex of proteins in the unreduced state. -
Figure 7.3.7 An example of a gel with uneven separation and staining. For optimal results, each sample lane in a gel should be loaded with the same amount of either sample or blank sample buffer so that the electrical characteristics of each lane are consistent. This prevents lane spreading (lane 10). Uneven staining can be minimized by gentle shaking in a large volume of staining solution during the staining process. Loss of high molecular-weight proteins and a general “fuzziness” in separation (lane 1) can result from the use of old or expired gels and buffers, or by protease activity in the sample. -
Figure 7.3.8 Separation of membrane proteins by 5.1% to 20.5% T polyacrylamide gradient SDS-PAGE. (%T = g acrylamide + g bisacrylamide/100 ml × 100). Approximately 30 µl of 1× SDS sample buffer containing 30 µg of Alaskan pea (Pisum sativum) membrane proteins were loaded in wells of a 14 × 14–cm, 0.75-mm-thick gel. Standard proteins were included in the lanes 1 and 10. The gel was run 15 hr at 4 mA. -
Figure 7.3.9 An example of vertical streaking. The vertical streaks of stained material are caused by skin flakes polymerized into the gel during gel preparation. When the dye front passes through during electrophoresis, the proteins in the skin flake solubilize and electrophorese, causing the tail of stained material. Filtering solutions and blowing out dust from the gel sandwich before polymerization will minimize these issues. The circular stain in the far right lane was caused by another source of protein: a small insect that dropped onto the gel during handling. -
Figure 7.3.10 An example of the effect of heating-related aggregation and endogenous proteases on the pattern of separated proteins. Lane 2 contains purified membrane proteins that were not heated in SDS sample buffer. The standard samples in lanes 1 and 4 were heated, as was the membrane sample in lane 3. Note that heating caused aggregation of the membrane protein, so that the proteins collected at the top of the separating gel. Unheated sample proteins (lane 2) show the major band of the ATPase ion transport catalytic subunit (arrow) otherwise lost using typical sample preparation and heating protocols. Heating the sample in SDS is typically used to denature the sample and inactivate proteases that are active in the SDS sample buffer; however, this can lead to aggregation of membrane proteins. Eliminating heating and using protease inhibitors instead can prevent heat-induced aggregation. Protease inhibitors added to the SDS sample buffer gave a slightly higher molecular weight for several proteins in the sample, indicating degradation by endogenous proteases (not shown). The center line indicates two portions of the same gel that were cut and pasted together for ease of comparison.
Videos
Literature Cited
| Literature Cited | |
| Adams, L.D. and Gallagher, S. 2004. Two-dimensional gel electrophoresis. Curr. Protoc. Mol. Biol. 67:10.4.1-10.4.23. | |
| 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. 1999. One-dimensional electrophoresis using nondenaturing conditions. Curr. Protoc. Mol. Biol. 47:10.2B.1-10.2B.11. | |
| Gallagher, S.R. 2006. One-dimensional SDS gel electrophoresis of proteins. Curr. Protoc. Mol. Biol. 75:10.2A.1-10.2A.37. | |
| 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. | |
| Jovin, T.M. 1973. I. Steady-state moving-boundary systems formed by different electrolyte combinations. Biochemistry 12:871-879. | |
| 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. | |
| Ornstein, L. 1964. Disc electrophoresis. I. Background and theory. Ann. N.Y. Acad. Sci. 121:321-349. | |
| Ploegh, H.L. 1995. One-dimensional isoelectric focusing of proteins in slab gels. Curr. Protoc. Protein Sci. 0:10.2.1-10.2.8. | |
| 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. | |
| Scopes, R.K. 1995. Overview of protein purification and characterization. Curr. Protoc. Protein Sci. 0:1.1.1-1.1.6. | |
| 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. | |
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An excellent book describing gel electrophoresis of proteins. | |
