Analysis of Protein Molecular Weight by SDS-PAGE

Protein molecular weight determination is fundamental in proteomics, providing insights into protein structure, function, and interactions. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) is a widely used method for analyzing protein molecular weight.

 

SDS-PAGE is a type of electrophoresis that separates proteins based on their molecular weight. The technique involves the denaturation of proteins using SDS, an anionic detergent, which imparts a uniform negative charge to the proteins. This uniform charge-to-mass ratio ensures that the proteins are separated solely based on their size during electrophoresis.

 

Key Components and Reagents of SDS-PAGE

1. SDS

Sodium dodecyl sulfate (SDS) denatures proteins by disrupting non-covalent bonds, ensuring that the proteins unfold into linear chains and acquire a negative charge proportional to their length.

 

2. Polyacrylamide Gel

The gel matrix, typically composed of acrylamide and bis-acrylamide, acts as a molecular sieve, allowing smaller proteins to migrate faster than larger ones.

 

3. Running Buffer

This buffer maintains the pH and ionic strength, ensuring consistent electrophoresis conditions.

 

4. Protein Standards

Known molecular weight markers are run alongside the samples to provide reference points for estimating the molecular weights of the sample proteins.

 

Process of Analyzing Protein Molecular Weight by SDS-PAGE

1. Sample Preparation

The protein samples are mixed with an SDS-containing sample buffer, which often includes a reducing agent like β-mercaptoethanol or dithiothreitol (DTT) to break disulfide bonds, a tracking dye, and a buffer to maintain the pH. The mixture is then heated to ensure complete denaturation.

 

2. Gel Casting and Electrophoresis

(1) Gel Casting: The polyacrylamide gel is cast between two glass plates, with a stacking gel on top of a resolving gel. The stacking gel has a lower acrylamide concentration and pH, allowing proteins to concentrate before entering the resolving gel, which has a higher acrylamide concentration and pH optimized for protein separation.

(2) Sample Loading: Denatured protein samples and molecular weight standards are loaded into the wells of the stacking gel.

(3) Electrophoresis: An electric field is applied, causing the negatively charged proteins to migrate through the gel towards the anode. Smaller proteins move faster through the gel matrix, resulting in size-based separation.

 

3. Visualization

After electrophoresis, the separated proteins are visualized using staining methods such as Coomassie Brilliant Blue or silver staining. The resulting bands correspond to proteins of different molecular weights.

 

4. Molecular Weight Estimation

A protein molecular weight marker (or ladder) with known protein sizes is run alongside the samples. By comparing the migration distance of the unknown proteins to the marker, the molecular weight of the proteins can be estimated.