SDS-polyacrylamide gel electrophoresis

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SDS-polyacrylamide gel electrophoresis is a widely used technique in biochemistry and molecular biology for separating proteins based on their size. This method is essential for determining the relative molecular mass of proteins and understanding their structural properties.

Purpose:

(1) To understand the basic principle behind SDS-polyacrylamide gel electrophoresis.

(2) To master the technique of determining the relative molecular mass of unknown proteins using this method.

Experimental Principle:

Sodium dodecyl sulfate (SDS) is an anionic detergent that denatures proteins by breaking hydrogen and hydrophobic bonds. It binds to the protein molecules, giving them a uniform negative charge. This masks the original charge differences between proteins, allowing electrophoretic mobility to depend primarily on molecular size. By comparing the migration distance of unknown proteins with that of standard proteins, the relative molecular mass can be estimated from a standard curve.

Materials and Reagents:

Reagents include 30% separation gel stock solution, 10% stacking gel stock solution, Tris-HCl buffers at different pHs, electrophoresis buffer, sample loading buffer, Coomassie Brilliant Blue R-250 for staining, and decolorization solution. Equipment includes a vertical electrophoresis tank, pipettes, glass plates, and syringes.

Procedure:

The process involves setting up the electrophoresis apparatus, preparing the separation and stacking gels, loading samples, running the gel, staining, and analyzing the results. After electrophoresis, the gel is stained with Coomassie Blue, and protein bands are visualized. The relative mobility of each band is measured, and a standard curve is plotted to estimate the molecular mass of the unknown proteins.

Precautions:

It is crucial to use a separate standard curve for each experiment. SDS may interfere with dye binding, so pre-treatment with decolorizing solution is recommended. For aqueous samples, the concentration should be adjusted accordingly before loading.

This technique is fundamental in protein analysis and plays a key role in research, quality control, and diagnostics in biotechnology and life sciences.

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