Biochem Final Protein Purification

Question 1

separation relies of physical and chemical properties such as

Answer 1

charge, size, affinity for a ligand, solubility and hydrophobicity
prosthetic groups can be used to isolate proteins

Question 2

benefits of chromatography

Answer 2

preparative separation in which the proteins can remain folded

Question 3

physical methods of breaking cells open

Answer 3

sonication - machine makes frequency to break open cells
french press - pressure breaks cells
grinding - cryo grinding with liquid nitrogen, machine shakes the frozen cells
followed by centrifugation

Question 4

chemical method of breaking open cells

Answer 4

lysis with detergent
1) native preparation - non charged detergent doesn’t denature 2) denatured prep - charged detergent, like SDS denatures proteins
followed by centrifugation

Question 5

bottom of solution that has been centrifuged

Answer 5

pellet - the bottom of the precipitate containing proteins

Question 6

sedimentation analysis by ultracentrifugation

Answer 6

sugar and glycerol used to make gradient - artificial gravitational field
create a vacuum and spin sample very fast
proteins will stop where they are the same density as the gradient (depending on shape as well)

Question 7

column chromatography

Answer 7

Detector detects proteins and separates out into different tubes as they are pushed through which separate based on mass properties and affinity for the matrix
lower affinity for matrix will wash out sooner, higher affinity will wash out last

Question 8

ion exchange separation

Answer 8

resin gradient in tube
protein mix on top, affinity to negatively charged resin will effect how quickly proteins move through the tube (repulsed will move faster)
slowly increase pH to ensure all proteins move through

Question 9

size exclusion separation

Answer 9

porous channels like a labyrinth
largest molecules move faster by taking shorter path

Question 10

separation by affinity

Answer 10

protein that binds to a certain ligand, antibody or protein
separate out and detach from ligand

Question 11

SDS PAGE

Answer 11

SDS detergent followed by polyacrylamide gel electrophoresis
separate proteins by size
Using electric field to pull molecules across gel matrix, larger mass will migrate more slowly, smaller will pass quickly

Question 12

how does SDS work?
how is it used?

Answer 12

sodium dodecyl sulfate
hydrophobic tail, charged head denatures proteins and then forms a micelle around it
gives negative charge to proteins
sometimes used with heat to help denature protein

used to analyze proteins based on molecular weight “size”
can be used to check you isolated the right protein

Question 13

aromatic amino acid detection

Answer 13

aromatic amino acids absorb light in the UV range 275-280 nm
strongest with tryptophan and tyrosine (phenylalanine less strong)
good for concentration of proteins with known sequences

Question 14

what to use to denature disulfide bonds for PAGE?

Answer 14

DDT or 2-mercaptoethanol
added following SDS
this is considered reducing PAGE

Question 15

native vs SDS page

Answer 15

SDS page much clearer migration results than native page (no denaturation of proteins)

Question 16

isoelectric focusing

Answer 16

used to determine pI of protein
protein added to one side of gel strip with immobilized pH gradient
protein will move until it reaches its pI (low pH, positive charge and vice versa)

Question 17

2D electrophoresis

Answer 17

combo isoelectric focusing 1st and SDS PAGE 2nd, makes a plot
x axis pI (increasing to decreasing) and y axis is low to high molecular weight

Question 18

why does specific activity of purified protein increase as overall protein activity decreases?

Answer 18

You start with a lot of cells, as you purify, number of proteins decreases. Increasing activity of single substrate because a lot of it is isolated, rather than a mix of proteins all together. Decreases impurities.