Lecture 13 Flashcards Preview

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Flashcards in Lecture 13 Deck (30):
1

Enzymes can be quantified by..

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

2

1 unit (U) of enzyme will catalyze

1umol in 1 min

3

Activity in a reaction equals...

U/uL or U/mL

4

specific activity equals...

activity/[protein]
-U/mg or U/ug

5

bimolecular purification

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

6

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

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

7

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

pH changes, heat denaturation, oxidation, proteolysis

8

how to avoid pH changes

extract/keep in buffered solutions

9

how to avoid heat denaturation

keep sample cold and heat only when needed

10

how to avoid oxidation

reducing agents can be added to prevent spontaneous crosslinking

11

how to avoid proteolysis

lower temp or add protease inhibitors such as EDTA

12

assays should be..

-sensitive
-specific
-rapid
-quantitative

13

yield

total activity after purification / total activity before purification

14

purity

specific activity after / specific activity before

15

purification by properties

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

16

ionic charge

– Ion exchange chromatography
– Electrophoresis
– Isoelectric focusing

17

polarity

reverse phase and hydrophobic chromatography

18

size

-dialysis
-size-exclusion chromatography
-gel electrophoresis

19

binding specificity

affinity chromatography

20

centrifugation

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

21

salting out

-common and reversible
-protein molecules with insufficient hydration will aggregate and lose solubility

22

dialysis

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

23

chromatography

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

24

stationary phase

a substance that the compounds to be separated pass
by or interact with

25

mobile phase

the carrier for the compounds to be separated

26

chromatography basic steps

-pouring
-packing
-loading
-running/eluting
-collecting

27

Vt

volume of column; pir^2h

28

Vo

volume outside beads

29

Vi

volume inside beads

30

Ve

volume at which a sample is eluted