Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis

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BACKGROUND

SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis, is a technique used in biochemistry and molecular biology to separate proteins according to their electrophoretic mobility (a function of length of polypeptide chain or molecular weight as well as higher order protein folding, posttranslational modifications and other factors).¹

The solution of proteins to be analyzed is first mixed with SDS, an anionic detergent which denatures secondary and non–disulfide–linked tertiary structures and dithiothreitol (DTT) or 2- mercaptoethanol (beta-Mercaptoethanol/BME) which reduces the disulfide bonds, and applies a negative charge to each protein in proportion to its mass.^1,2 Without SDS, different proteins with similar molecular weights would migrate differently due to differences in folding, as differences in folding patterns would cause some proteins to better fit through the gel matrix than others. Adding SDS solves this problem, as it linearizes the proteins so that they may be separated strictly by length (primary structure, or number of amino acids). The SDS binds to the protein in a ratio of approximately 1.4 g SDS per 1.0 g protein (although binding ratios can vary from 1.1 g SDS/g protein), giving an approximately uniform mass:charge ratio for most proteins, so that the distance of migration through the gel can be assumed to be directly related to only the size of the protein. A tracking dye may be added to the protein solution to allow the experiment or to track the progress of the protein solution through the gel during the electrophoretic run.³

The denatured proteins are subsequently applied to one end of a layer of polyacrylamide gel sub- merged in a suitable buffer. An electric current is applied across the gel, causing the negatively- charged proteins to migrate across the gel. Depending on their size, each protein will move differently through the gel matrix: short proteins will more easily fit through the pores in the gel, while larger ones will have more difficulty.^3,4

After a set amount of time (usually a few hours- though this depends on the voltage applied across the gel; higher voltages run faster but tend to produce somewhat poorer resolution), the proteins will have differentially migrated based on their size; smaller proteins will have traveled farther down the gel, while larger ones will have remained closer to the point of origin. Thus proteins may be separated roughly according to size (and therefore, molecular weight).^1,4 Following electrophoresis, the gel may be stained (most commonly with Coomassie Brilliant Blue or silver stain), allowing visualization of the separated proteins, or processed further (e.g. Western blot). After staining, different proteins will appear as distinct bands within the gel. It is common to run “marker proteins” of known molecular weight in a separate lane in the gel, in order to calibrate the gel and determine the weight of unknown proteins by comparing the distance traveled relative to the marker. Gel electrophoresis is usually the first choice as an assay of protein purity due to its reliability and ease. The presence of SDS and the denaturing step causes proteins to be separated solely based on size.

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REQUIREMENTS

Materials

30% Acrylamide/bis (29:1) solution;²

4 x Resolving gel buffer: 1.5 M Tris-HCl (pH 8.8), 0.4% SDS;

4 x Stacking gel buffer: 0.5 M Tris-HCl (pH 6.8), 0.4% SDS;

1 x Elecrtophoresis buffer: 25 mM Tris-HCl (pH 8.3), 250 mM glycine, 0.1% SDS.

3 x protein loading buffer: 0.19 M Tris-HCl (pH 8.0), 3% glycerol, 6% SDS, 300 mM DTT, and 0.25% bromophhenol blue;

10% Ammonium persulfate (APS);

TEMED;

Gel staining buffer: 1 g of Coomassie Brilliant Blue dissolved in 400 ml of solution con- taining 45% methanol, 45% H₂O, and 10% acetic acid;

Destaining buffer: 25 % methanol, 10% acetic acid, and 65% H₂O;

Gel Drying Buffer: 40% ethanol, 4% glycerol, and 56% H₂O.

Protein samples.

Protein standards.

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PROCEDURE

Prepare 40 ml of 15% resolving gel solution as following:²

First assemble the gel caster, and add the 15% gel mix to gel caster. Overlay the solution gently with water or 1-butanol saturated with H₂O, and allow to be polymerized. You should see the gel-H₂O interface disappear and then reappear when the gel is polymerized;

Prepare 20 ml of 5% stacking gel solution as following:²

Add the gel mix to the gel caster, and insert the comb to the gel solution; you should wait until gel is fully polymerized. Once the gel is polymerized, the comb can be removed gently. Then assemble the gel into the electrophoresis apparatus. For loading of samples; mix two volumes of your samples with 1 volume of 3 x sample loading buffer, and heat the samples over 90⁰C for 5 min, and load 20 µl of your samples to each well of the gel. Also add the protein standards in two lanes on each gel. Running of gel requires power supply at 100-150 V until the dye has reached the top of the resolving gel, and then increase the power into 200 V. When the dye reaches the bottom of the resolving gel, turn off the power supplier. Disassemble the gel apparatus, and stain the gel for about one hour at Gel staining buffer. Destain your gel at the destaining buffer until the protein bands are clearly seen in the gel, and dry the gel if it is possible. The molecular weight of the protein can be determined by measuring the migration of proteins and the dye, and calculate the mobility of each protein by using the following equation. Plot the mobility versus log value of molecular weight of the stand proteins, and then determine the molecular weight of the protein sample:

Mobility = (Distance of the protein migrated)/ (Distance of the dye migrated)

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CONCLUSION

From the above experiment of SDS (sodium dodecyl sulphate) electrophoresis it can be concluded that the protein sample having higher molecular weight are retained at the top of the gel and rest of the proteins shows band according to their molecular weight.

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REFERENCES

1. www.creativebiomart.net/blog/principle-and-procedure
   
2. Biochemistry lab manual by Fenfei Leng Florida International University Department of Chemistry Revised, spring 2009; pp 27-29 CHM-4304L
   
3. J., Ninfa, Alexander; P., Ballou, David (2004). Fundamental laboratory approaches for biochemistry and biotechnology., Wiley.
   
4. Sambrook j, Russel DW(2001). Molecular cloning: A laboratory Manual 3^rd Ed. Cold spring harbor lab press.
   
5. Practical biotechnology (Methods and Protocols ) ; S Janarthanan & S Vincent .