Exploring Proteins Flashcards
How to make more protein sample.
- Introduce DNA into host cell like bacterial e.coli.
- Lyse bacteria cells to extract the protein.
What is SDS-PAGE?
monitors protein purification, separates protein by size, determines RMN.
AKA Sodium dodecyl sulphate polyacrylamide gel electrophoresis.
What is SDS?
AKA sodium dodecyl sulphate.
An anionic detergent that denatures proteins and gives a negative charge to the denatured polypeptide.
Protein moves in the direction of positive anode in an electric field.
What is SDS?
AKA sodium dodecyl sulphate.
An anionic detergent that denatures proteins and gives a negative charge to the denatured polypeptide.
Protein moves in the direction of positive anode in an electric field.
What is PA?
AKA polyacrylamide. Gel of crossed linked monomers of acrylamide. High conc. of acrylamide means more closely packed PA will be.
What is PG?
AKA gel electrophoresis.
Bigger protein, more difficult to travel through PA. Protein filtered out on its size.
What is salting out?
Salt competes with protein to be soluble in water. More salt, less water available for protein to form H-bonds.
Electrostatic attraction between low conc. of salt and protein, binding makes protein soluble.
How is the salt removed?
Conc.protein is placed in dialysis tube, sealed then suspended in contain of buffer that the protein must be replaced with.
How to isolate protein without denaturing.
Chromatography called gel filtration. After salting out and being in a buffer.
Collins of resin, made from small porous beads which allows protein to fall through. Bigger ones stay amongst beads will smaller ones fall to the bottom.
How can protein be isolated using their charges?
After salting out and gel filtration, ion exchange would happen. Last step of purification.
Explain ion exchange.
Protein mix into resin. Negative protein will bind to positive resin, positive protein won’t and will fall through.
How to determine structure of protein.
Using NMR and X-ray crystallography
How to determine physio-chem properties of proteins.
Amino acid sequence, isoelectric point, similarity to other proteins.
How to determine enzymatic properties of protein.
Rate of catalytic activity. Inhibition.
Explain what is affinity chromatography .
Eg. Antibody binds to human amylase. Antibody covalently link to resin which can be used to specifically bind to amylase. All other proteins will fall through.
