Gel Electrophoresis

Principle

• Separate by molecular weight

• Smaller molecular proteins move faster

Key Chemicals - SDS

Sodium dodecyl sulfate

polyacrylamide gel electrophoresis

SDS is something that is added into shampoos, detergent

Key Chemicals - β-mercaptoethanol/DTT

• Reducing compound that breaks disulfide bonds 

• Reducing agent that denature the protein to a rod shape

Key Chemicals - Acrylamide

• Monomer→ polymer gel to ensure there is no toxicity

Sample preparation

Buffer: glycerol, SDS, Tris-HCl 6.8, β-mercaptoethanol (reducing agent), bromophenol blue (dye)

Reducing Conditions

adding agents like DTT or β-mercaptoethanol to break disulfide bonds, unfolding proteins and separating them into individual subunits, revealing their true molecular weight (size)

Non-reducing conditions

separates proteins without breaking their disulfide bonds, revealing their native or complex structure

Running gels

• Tris base, glycine, SDS

Staining solution

Coomassie blue, methanol, glacial acetic acid

Destaining solution

Methanol, glacial acetic acid