SDS-PAGE and Protein Electrophoresis

SDS-PAGE

• SDS-PAGE is a technique used to separate proteins based on their molecular weights
• Proteins are negatively charged in SDS-PAGE, meaning they migrate towards the positive electrode
• SDS (Sodium Dodecyl Sulfate) denatures proteins and gives them a uniform negative charge, eliminating the effect of charge and structure
• Proteins are separated solely based on differences in their molecular weights
• Molecular weight of a protein is the sum of masses of its constituent amino acids
• The separation is achieved within a polyacrylamide gel with a mesh size that is suitable for the separation of proteins of typical size.

Protein Electrophoresis

• Electrophoresis is a technique that separates macromolecules (proteins) in a mixture using an electric current
• In this process, proteins migrate through the gel, separated by their molecular weight
• The polyacrylamide gel matrix acts as a molecular sieve, with smaller proteins moving through the pores faster than larger proteins
• SDS-denatured proteins migrate based on their size rather than other factors.

Applications of SDS-PAGE

• Protein Purification: Evaluating the purity of protein samples
• Molecular Weight Estimation: Determining the protein's molecular weight
• Protein Identification: Identifying proteins in complex mixtures
• Comparative Analysis: Comparing the expression levels of proteins under different conditions

Preparing Proteins for SDS-PAGE

• Denaturing proteins, before electrophoresis, is often necessary for a conclusive result
• Heat destroys hydrogen bonds while reducing agents, such as $\beta$-mercaptoethanol (BME), break disulfide bonds
• This linearizes the proteins, making them uniform, and only size-dependent

Materials Required for SDS-PAGE

• Gel apparatus and electrophoresis apparatus
• 30% acrylamide/0.8% bis-acrylamide stock solution
• 2.5x separating gel buffer
• 5x stacking gel buffer
• TEMED (Tetramethylethylenediamine)
• 10% ammonium persulfate
• Butane-1,2-diol
• 5x electrophoresis buffer
• 5x SDS sample buffer
• Protein samples
• Protein Ladder (for size determination)

Preparation of Polyacrylamide Gel

• Gel casting
• Resolving gel
• Stacking gel
• Isopropanol is placed atop the resolving gel to prevent air bubbles and make the resolving gel surface smoother
• The stacking gel has lower acrylamide percentage, different pH and ionic composition compared to the resolving gel
• A comb is placed atop the gel to create “wells” where protein samples can be loaded
• Different amounts of acrylamide and bis-acrylamide form gels with varying pore sizes suitable for different protein sizes

Run the SDS-PAGE

• After casting the gel, samples are loaded into wells
• An electric current is passed through the gel, causing the negatively charged proteins to migrate towards the positive electrode

Running Buffer

• Maintains the correct pH, facilitates the current flow and electrophoretic separation
• Three buffers are usually used in SDS-PAGE: buffer for casting the gel, buffer for sample preparation and buffer for running the gel

Loading Samples

• Each sample is loaded into a well in the gel, using SDS-PAGE loading buffer
• The loading buffer contains SDS and usually also glycerol (this increases the density of the sample) and a tracking dye such as bromophenol blue, which helps to monitor the migration of the sample through the gel
• Samples should be heated prior to loading to ensure proteins are denatured and linear

Glycerol

• Increases the density of the sample so the sample drops to the bottom of the well, preventing spillover

Bromophenol Blue

• Dye used to track the migration of the sample, and thus can be used to determine migration distances
• The dye is negatively charged and will migrate towards the positive electrode, helping determine when the separation is complete

Protein Ladder

• Contains protein samples of known molecular weights
• Used to determine the estimated size/weight of the unknown samples
• Each rung of the ladder corresponds to a polypeptide of a specific size.

Visualization of the Gel

• Coomassie Blue Staining- Visualizing and identifying separated protein bands
• Silver Staining- Sensitive, able to detect as little as 2-5ng of protein per band
• Gels are incubated in a solution that stains the separated proteins, making them visible

Interpreting Results

• The separated proteins will form bands in the gel
• The distance traveled by a protein can be used to estimate its molecular weight
• Comparing the migration distance of the sample protein to the known molecular weight markers in the protein ladder can determine the protein’s size