Lecture 13

Question 1

Enzymes can be quantified by..

Answer 1

comparing the amount needed to complete a set amount of reaction in a set period of time

Question 2

1 unit (U) of enzyme will catalyze

Answer 2

1umol in 1 min

Question 3

Activity in a reaction equals…

Answer 3

U/uL or U/mL

Question 4

specific activity equals…

Answer 4

activity/[protein]

-U/mg or U/ug

Question 5

bimolecular purification

Answer 5

breaking cells apart and isolating organelles, proteins, DNA, RNA & lipids for individual studies

Question 6

(Protein Extraction) Cells, tissue, or organisms are broken apart by…

Answer 6

• sonication
• french press
• osmotic shock
• digestion
• detergents
• homogenization

Question 7

Proteins removed from a cell may become degraded by which non-physiological conditions?

Answer 7

pH changes, heat denaturation, oxidation, proteolysis

Question 8

how to avoid pH changes

Answer 8

extract/keep in buffered solutions

Question 9

how to avoid heat denaturation

Answer 9

keep sample cold and heat only when needed

Question 10

how to avoid oxidation

Answer 10

reducing agents can be added to prevent spontaneous crosslinking

Question 11

how to avoid proteolysis

Answer 11

lower temp or add protease inhibitors such as EDTA

Question 12

assays should be..

Answer 12

• sensitive
• specific
• rapid
• quantitative

Question 13

yield

Answer 13

total activity after purification / total activity before purification

Question 14

purity

Answer 14

specific activity after / specific activity before

Question 15

purification by properties

Answer 15

• solubility (salting out)
• ionic charge
• polarity
• size
• binding specificity

Question 16

ionic charge

Answer 16

– Ion exchange chromatography
– Electrophoresis
– Isoelectric focusing

Question 17

polarity

Answer 17

reverse phase and hydrophobic chromatography

Question 18

size

Answer 18

• dialysis
• size-exclusion chromatography
• gel electrophoresis

Question 19

binding specificity

Answer 19

affinity chromatography

Question 20

centrifugation

Answer 20

if the protein is soluble it can be partially purified by removing larger/denser contaminants

Question 21

salting out

Answer 21

• common and reversible

- protein molecules with insufficient hydration will aggregate and lose solubility

Question 22

dialysis

Answer 22

desalting; dialysis tubing has pores with a specific molecular weight cut-off that allow smaller molecules (salt) to pass

Question 23

chromatography

Answer 23

Techniques to separate mixtures based on physical properties such as size or charge; mixed sample needs to have variable affinity

Question 24

stationary phase

Answer 24

a substance that the compounds to be separated pass

by or interact with

Question 25

mobile phase

Answer 25

the carrier for the compounds to be separated

Question 26

chromatography basic steps

Answer 26

• pouring
• packing
• loading
• running/eluting
• collecting

Question 27

Vt

Answer 27

volume of column; pir^2h

Question 28

Vo

Answer 28

volume outside beads

Question 29

Vi

Answer 29

volume inside beads

Question 30

Ve

Answer 30

volume at which a sample is eluted