Lab

Question 1

enzyme activity

Answer 1

in units per ml (units ml-1)
where 1 unit of activity is the amount of enzyme needed to convert 1 umole of substrate to product in a 1 minute

Question 2

specific activity =

Answer 2

enzyme activity/protein conc in: units mg-1

Question 3

total activity

Answer 3

enzyme activity multiplied by the volume of the sample
units ml-1 x ml –> leaving just units
the total number of units in that sample at that stage

Question 4

% yield

Answer 4

(total activity after / total activity before) x 100%

Question 5

purification factor

Answer 5

(specific activity after / specific activity before) x 100%

Question 6

gel filtration (size exclusion)

Answer 6

different sized beads, smaller ones take longer to travel through

Question 7

how do you monitor the purification process

Answer 7

SDS page between each step
- activity measurements, make sure the protein is working not unfolded etc.

Question 8

anion exchange chromatography

Answer 8

resin with positively charged functional groups to capture negatively charged proteins

Question 9

cation exchange chromatography

Answer 9

resin with negatively charged functional groups to capture positively charged proteins

Question 10

hydrophobic interaction chromatography

Answer 10

water “removed” using high [salt] proteins would rather bind to the chromatographic matrix than stay in solution
elute with lowered [salt]

Question 11

affinity chromatography

Answer 11

most powerful chromatographic method - based on specific binding to the matrix
eg. His6

Question 12

dye chromatography

Answer 12

type of pseudo-affinity chromatography
show
dyes show certain structural similarities to cofactors such as NAD(H) and NADP(H)