Analysis of Proteins

Question 1

what are proteins fractioned by ?

Answer 1

by column chromatography

Question 2

proteins can be separated according to their:

Answer 2

1. Charge (ion-exchange chromatography)
2. Hydrophobicity (hydrophobic chromatography)
3. Size (gel-filtration chromatography)
4. Ability to bind to particular small molecules or to other macromolecules (affinity chromatography)

Question 3

what happens to proteins in SDS plyacrylamide-gel eletrophoresis (SDS-PAGE) ?

Answer 3

• An electric field applied to a solution containing a protein molecule which cause protein to migrate at a rate that depend on its net charge and on its size and shape.
• Uses a highly cross-linked gel of polyacrylamide as the inert matrix through which the proteins migrate

Question 3

what happens to proteins in SDS plyacrylamide-gel eletrophoresis (SDS-PAGE) ?

Answer 3

• An electric field applied to a solution containing a protein molecule which cause protein to migrate at a rate that depend on its net charge and on its size and shape.
• Uses a highly cross-linked gel of polyacrylamide as the inert matrix through which the proteins migrate

Question 4

How does SDS-PAGE work ?

Answer 4

1. Proteins are dissolved in a solution that includes negatively charged detergent, Sodium Dodecyl Sulphate
2. Detergent bind to hydrophobic regions of the protein moleules, causing them to unfold into extented polypeptide chains
3. Invdividual proteins are released from their association with other proteins or lipids.
4. They now freely soluble in the detergent solution
5. Reducing agent (e.g.β-mercaptoethanol) is added to break S-S linkages in proteins, so that multi-subunit proteins can be analysed separatly

Question 5

steps of Western Blotting

Answer 5

1. Take cellular proteins from different conditions
2. unfold & coat with negative charge with SDS
3. Put the through gel electrophotrsis which separates proteins by size
4. Move proteins to a memebrane (TRANFER BLOT)
5. Coat the free memebrane with a genetic protein (BLOCK)
6. Binding specific portein you are looking for (PRIMARY ANTIBODY BINDING)
7. BINDS PRIMARY ANTIBODY and lets you detect it
8. WASH
9. See how much of the specific protein is there (VISUALIZE)

Question 6

How does ELISA work ?
Enzyme-linked Immunosorbent Assay

Answer 6

1. uses a solid-pahse of enzyme immunoassay (EIA) to detect presence of a ligand (common protein) in a liquid sampke using antibodies directed against the protein to be measured