Using SDS-PAGE to assess the purification of a protein.
Techniques to Master:
Denaturing Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE)
A. SDS-PAGE separates proteins in a mixture. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) is a technique used by biochemists to analyze and separate proteins in a mixture. The technique takes advantage of the fact that a protein with a net charge will move in an electric field (electrophoresis). The proteins are applied to a vertical gel that acts as a molecular sieve. As the proteins are pulled through the gel by the electric field, they interact with the porous gel such that smaller proteins travel farther through the gel than larger proteins, which are retarded by the solid gel matrix.
B. Sample buffer and boiling denatures the proteins prior to analysis. Prior to analysis, the proteins are mixed with a sample buffer and boiled. The sample buffer contains sodium dodecyl sulfate (SDS) and a reducing agent (such as beta-mercaptoethanol or dithiothreitol). SDS is a negatively charged detergent that disrupts the noncovalent interactions of the proteins (Figure 1). SDS binds to the main chains of the proteins at a ratio of about 1 SDS per 2 amino acids . In this way, all proteins are coated with an abundance of negative charges to facilitate their movement from the negative pole (anode) toward the positive pole (cathode) during the electrophoresis. Moreover, the charge on the protein becomes relatively proportional to the number of amino acids (or molecular weight) of the protein. The reducing agent reduces any disulfide bonds. The sample buffer and boiling effectively denature the proteins in the sample. This reduces the complications of secondary, tertiary, or quaternary structure that would affect mobility through the gel. Glycerol is also added to the sample buffer. Glycerol is denser than water and ensures that the solution does not diffuse out of the sample wells and into the running buffer.
C. The gel is formed by crosslinking acrylamides. The electrophoresis medium of choice for proteins is polyacrylamide - formed by crosslinking acrylamide and methylenebisacrylamide. Precast gels can be purchased or gels can be cast manually prior to use. In either case, acrylamide and bisacrylamide are pre-mixed, then combined with buffer, SDS, ammonium persulfate, and tetramethylethylenediamine (TEMED). A radical polymerization reaction is initiated by addition of persulfate. Persulfate is converted to sulfate radical and causes the polymerization of acrylamide with occasional cross-links by bis-acrylamide (Figure 2). In practice, upon addition of the last two reagents the liquid is quickly poured into the narrow space between two glass or plastic plates for polymerization. The gels are run in a vertical position using a running buffer system that is at a high pH and contains SDS.
D. A discontinuous SDS-PAGE system increases resolution. To increase resolution a discontinuous SDS-PAGE system can be used (Figure 3) . In discontinuous SDS-PAGE the acrylamide gel consists of two parts: the bottom is the separator, or resolving gel, because this is where the separation based on size occurs. The upper gel is the stacking gel, which causes all the proteins to become concentrated into a very tight boundary, due to this gel having a lower pH (6.8) than the separator and the running buffer (pH 8.8). The high pH of the separator and the running buffer causes all aspartates, glutamates, histidines, and lysines in the proteins to be deprotonated, helping to make the proteins as negatively charged as possible. The upper gel also has a larger pore size due to the use of a lower percentage of acrylamide, which allows the proteins to migrate faster until the more dense resolving gel is reached.
The percent acrylamide of the resolving gel is chosen based on the known or predicted size of the protein under investigation .The best information about the size and purity of a protein comes if the protein migrates to the middle of the gel. Different percentages of acrylamide are achieved by including different ratios of acrylamide to water in the gel reaction (Table 1).
The purpose of the stacking gel is to concentrate the protein sample into a tight band. This ensures that proteins enter the resolving gel from the same starting point. The stacking gel is at pH 6.8 (note that running buffer and resolving gel are at pH 8.8). As the proteins travel through the stacking gel, their ionization state continuously changes: at the lower pH (pH 6.8), proteins become protonated and slow down, and at higher pH (pH 8.8 of the running buffer), they become deprotonated and speed up. This happens over and over, and the result is that all the proteins concentrate into a single tight band at the boundary between stacking and resolving gels. The polyacrylamide concentration in the stacking gel is low (~5%) to allow the protein concentrating process to affect every protein in the sample, regardless of protein size.
E. Estimating the molecular weight of a protein. Once the proteins move into the resolving gel, their mobility is dependent upon their size. The size of the protein of interest can be estimated through comparison with the migration of proteins of known molecular weight. At least one well of a gel should be loaded with a protein molecular weight standard, which is a mixture of proteins of known molecular weight. Such standards are usually purchased premade. After the gel is stained, a standard curve can be prepared that will allow the molecular weight of the protein of interest to be estimated (Figure 4).
F. Visualizing the proteins in the gel via staining. Once the proteins have been run on the gel, they must be visualized. While many stains are available, an easy and relatively sensitive method is the Coomassie Blue Staining method 4. Under acidic conditions, the dye interacts with positively-charged amino acids as well as aromatic amino acids. The acid in the stain fixes the proteins in the gel so that they don’t diffuse out. After the staining is complete a destaining solution is added to remove excess dye from the gel, this step minimizes the background and increases the contrast (Figure 5). There are some commercial stains available, such as InstantBlue, that do not require a destaining step.
With staining, the more intense/dense the band in the gel, the more protein is present. Often just observing the pattern of blue bands and their intensity and position is good enough for the purposes of the experiment. Other times it may be desirable to estimate relative concentrations of proteins in the bands, and this can be performed using a densitometry scanner and program.
Purpose: SDS-PAGE will be used to verify the purity of the protein of interest. Samples from different steps along the purification pathway will be analyzed on the gel. A “pure” protein should display a single band on the gel after staining. (Note that multi-subunit proteins may show more than a single band since the sample buffer protocol destroys quaternary structure.) The protein of interest should also migrate at the expected molecular weight when compared to the molecular weight standards.
Safety Considerations: Consult MSDS and bottle labels for safe handling of reagents and stains.
Experimental Design Considerations: When planning an SDS-PAGE experiment, consider the questions that can be answered with this type of analysis. What samples should be collected during the induction and purification? How will the number of bands in a sample vary as the purification proceeds? How will the molecular weight markers help you determine if you’ve purified the correct protein? Ultimately you will be generating a figure of the results, so consider the best order for loading the samples in the wells.
Electrophoresis gel and apparatus
Heating device for denaturing proteins
Reagents & Solutions:
2X SDS Sample Buffer:
0.8 ml 1.5 M Tris-HCl pH 6.8
0.4 g SDS (weigh out in the hood)
1.5 ml 100% glycerol
0.4 ml 14.7 M β-mercaptoethanol (open in the hood)
2 mg bromophenol blue
Fill to 10 mL with deionized water
Store at 4°C or freeze aliquots
10X Running Buffer:
32 g Tris base
144 g glycine
10 g SDS
Fill to 1 liter with deionized water
A. Sample Preparation. Protein samples must be treated with SDS sample buffer and boiled before application to the gel. The final concentration of the SDS sample buffer loaded into the gel should be 1X. The recipe above makes a 2X SDS sample buffer. By mixing equal amounts of this with your sample, the final concentration will be 1X.
B. Running the gel. Prior to running the samples, the gel must be set up in an appropriate apparatus with 1X running buffer added. The recipe above is for a 10X running buffer. It must be diluted 1:10 prior to use.
C. Staining the gel.
Interpreting Results: There are three main things to look for as you interpret your SDS-PAGE results. First, is there a new or more dense band in the post-induction sample that was not there in the pre-induction sample? This will show that the induction was successful. Second, as the purification progresses are fewer bands present? This is an indication that unwanted proteins are being removed and your protein of interest is being enriched. Third, does the molecular weight of the protein of interest, as estimated by comparison to the molecular weight standards, match the anticipated molecular weight? If not, what could be the reason? There may be additional information that can be gleaned from a well-designed SDS-PAGE experiment.
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