Protein Purification

Question 1

Histidine is often in the active site of the protein, v good chance if you see one it’s around there

Answer 1

ya

Question 2

What does it mean if the cysteines are an odd number
• Disulfide bond involves 2 cysteines
• One cannot have a partner, so there will be a free sulfhydryl group
○ This causes a lot of problems
○ Important to know # of cysteines

Answer 2

ya

Question 3

amino acids Phe, Tyr, and Trp absorb light that peaks at 280 nm–if your protein lacks these residues, it will be almost invisible at 280 nm.

Answer 3

ya

Question 4

what are lectins?

Answer 4

• Bind specific carbohydrates

May be able to set up a purification scheme based on the presence of a lectin

Question 5

what are the four basic techniques to purify protein?

Answer 5

ammonium sulphate; ion exchange column, gel (size) exclusion; affinity column

Question 6

what is the prosthetic group of lipoproteins, and give an example

Answer 6

beta 1 lipoprotein of blood

Question 7

prosthetic group of glycoproteins and give an example

Answer 7

carbohydrates, IgG

Question 8

prosthetic group of phosphoproteins, example

Answer 8

phosphate groups, casein of milk

Question 9

Hemoprotein prosthetic group and example

Answer 9

heme (iron porphyrin), hemoglobin

Question 10

Flavoproteins PG, example

Answer 10

flavin nucleotides, succinate dehydrogenase

Question 11

metalloprotiens PG, examples

Answer 11

iron, ferritin; zinc, alcohol DH; calcium, calmodulin; molybdenum, dinitrogenase; copper, plastocyanin

Question 12

describe the ammonium sulphate technique

Answer 12

ammonium sulph is very soluble (can make a 6M or 7M solution); make high salt sol’ns, proteins ppt because the cations and anions from the salt attach to the proteins and neutralize the charge, so the proteins clump together;
–add amm. sulph. in increments; 20% sol’n, some proteins (closer to already uncharged) will ppt; spin, collect supernatant, assay resuspended pellet; go to 40%, spin, remove, assay, etc