Polyacrylamide Gel Electrophoresis

Electrophoresis

• Horizontal Agarose Gels• Agarose forms a gel or molecular sieve

that supports the movement of small materials in solution used for DNA

• Vertical Polyacrylamide Gels • Made of Polyacrylamide • Used for Protein molecular size, shape, charge • IEF electrophoresis• Western Blot technique

PAGE

• Native : Protein is prepared with little disturbance to the cellular material– Proteins are associated –Movement of samples through the gel can be

inconsistent• SDS : Sodium Dodecyl Sulfate Is a detergent – Protein coated with a negative charge in

proportion to its molecular weight– Denatures and unfolds protein– Reducing agents (DTT)break amino acid cross-links

P

Polyacrylamide Gel Creates tunnels in gel for molecules to move through

Equipment for Electrophoresis

SDS Polyacrylamide Electrophoresis

Uses for PAGE

• Separates proteins from each other– Proteins separated by size– Isoelectric point

• Determines– Molecular size of protein– Quantifies the amount present– Displays Impurities– Used in western blot assays by antigen interactions

Procedure in Short

LoadGe Place Buffer

Equip

Determine Molecular Weight

1. Run standard molecular weight markers on gel2. Run unknown protein on the same gel3. Create a graph of the mol wt versus distance molecule has moved4. Using the distance the unknown has moved

determine the molecular weight from graph