What are the reagents used in SDS-PAGE electrophoresis?

The reagents used in SDS-PAGE electrophoresis include: 

Sodium Dodecyl Sulfate (SDS)

SDS is included in the sample buffer preparation to give the protein a negative charge. Following β-Mercaptoethanol treatment, proteins adopt a linear structure, enabling SDS to uniformly impart a negative charge to all the proteins. This causes the proteins to migrate towards the positive electrode during electrophoresis, facilitating their separation by molecular size. Additionally, SDS is present in the gel to ensure proteins remain negatively charged throughout the process.

β-Mercaptoethanol / Dithiothreitol (DTT)

Disulfide bonds between polypeptide chains are key to maintaining a protein’s secondary structure. β-Mercaptoethanol disrupts these disulfide bonds, converting the S-S linkages into -SH, -SH groups, thus freeing the polypeptide chains. This treatment results in proteins being reduced to their primary structure, allowing them to be separated based solely on size during gel electrophoresis.

Bromophenol Blue (BPB)

Bromophenol blue serves as a tracking dye during electrophoresis. It imparts color to the proteins in the gel and is mixed with the sample solution. Due to its slight negative charge, it migrates towards the positive electrode ahead of the proteins, indicating the progression of protein migration.

Polyacrylamide Gel

Polyacrylamide gel is formed when the monomer acrylamide in water polymerizes with a small amount of a cross-linker such as N,N’-Methylenebisacrylamide. This co-polymerization creates a 3D network of interconnected acrylamide chains. 

Alternative cross-linkers that may be used include:

• N,N’-Diallyltartardiamide (DATD): A disruptable cross-linker that solubilizes the gel
• Piperazine Diacrylate (PDA): Reduces background silver staining in SDS-PAGE