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 gel 2. Run unknown protein on the same gel 3. Create a graph of the mol wt versus distance molecule has moved 4. Using the distance the unknown has moved determine the molecular weight from graph