protein separation and purification

Question 1

protein property: solubility

Answer 1

method: disrupt cell membranes and create crude extract

Question 2

protein property: solubility (2)

Answer 2

precipitation with ammonium sulfate (salting out)

Question 3

protein property: size/shape

Answer 3

method: size exclusion chromatography

Question 4

protein property: isoelectric point (charge)

Answer 4

method: ion exchange chromatography

Question 5

protein property: binding to small molecules

Answer 5

method: affinity chromatography

Question 6

sonicator

Answer 6

sound waves disrupt cell membranes

Question 7

french press

Answer 7

pressure disrupt cell membrane

Question 8

decant off supernatant with soluble protein produces what?

Answer 8

soluble lysate

Question 9

at low salt concentrations, the solubility of a protein often increases

Answer 9

salting in

Question 10

as the concentration of salt increases, the solubility of protein decreases until it precipitates

Answer 10

salting out

Question 11

why do we use ammonium sulfate to precipitate proteins?

Answer 11

has a larger effect on solubility than other salts and it is very soluble so that solutions can be made at high concentrations

Question 12

low [salt]

Answer 12

strong attractive force between proteins

Question 13

optimal [salt]

Answer 13

salt ions “shield” charges on proteins so weak attractive force

Question 14

high [salt]

Answer 14

ions compete for water allowing hydrophobic and charge interactions

Question 15

ion exchange chromatography

Answer 15

positively or negatively charged functional groups attached to resin

Question 16

cation exchanger(negatively charged resin)

Answer 16

binds cations

Question 17

anion exchanger (positively charged resin)

Answer 17

binds anions

Question 18

what factors affect ion exchange chromatography?

Answer 18

net charge on molecule (greater net charge, better protein binding), solution pH, ionic strength (small ions compete with proteins for binding to the column)

Question 19

size exclusion chromatography

Answer 19

separates molecules based on size/shape; resin matrix is porous with pores in a defined size range

Question 20

analytical technique for protein MW estimation

Answer 20

protein MW can be estimated from a plot of elution volumes vs log MW. protein standards are run to define dependence of elution volume on size, then unknown protein is compared to standards

Question 21

affinity chromatography

Answer 21

proteins bind to resin via specific interactions with ligands covalently bound to the resin

Question 22

Polyacrylamide Gel Electrophoresis (PAGE)

Answer 22

protein is denatured by and coated with sodium docecyl sulfate (SDS); 1 SDS molecule for every 2 AAs; each SDS molecule is negatively charged so that SDS charge overwhelms the protein charge & separation is dependent on mass

Question 23

how can protein pI can be determined?

Answer 23

gel containing a pH gradient; proteins will be moved thru gel by electric field until they reach a region of pH where pI=pH; called ISOELECTRIC FOCUSING

Question 24

isoelectric focusing and SDS gel electrophoresis can be combined to do what?

Answer 24

to separate proteins according to their mass and pI in this 2D method