Lecture 11 - biochemical analysis

Question 1

3 stages of protein purification

Answer 1

1. sample homgenisation = physical disruption
   of cells or tissues
2. differential centrifugation
3. separating proteins based on their properties e.g. solubility, charge etc.

Question 2

5 lysis methods

Answer 2

• mechanical - blender
• liquid homogenisation = forcing cell/tissue through narrow space in liquid medium e.g. french press
• sonication = high frq sound to shear cells
• freeze/thaw = repeating will shear cells though ice crystal formation
• manual grinding = mortar pestle

Question 3

what does centrifuging at 500xg 10mins form

Answer 3

pellet has nuclear fraction

Question 4

what does centrifuging at 10,000x g for 20mins form

Answer 4

mitochondrial fraction
(will include chloroplasts too if plant cell)

Question 5

what does centrifuging at 100,000x g 1hr form

Answer 5

microsomal fraction
- ribosomes, ER etc

Question 6

Equation for RCM and acceleration

Answer 6

RCM = a/9.8
A = w^2r
Remember radius is in metres

Question 7

how to fraction proteins based on solubility

Answer 7

can precipitate proteins with COMPETING SOLUTE
e.g. ammonium sulphate, acetone, polyethylene glycol
to

Question 8

what properties should competing solute have

Answer 8

Very soluble in water (up to 50% weight/volume)
Non-damaging to proteins in general
Easy to remove from the proteins afterwards
Not too viscous or dense
Cheap and pure

Question 9

how to work out free energy of transport

Answer 9

used in the context of dialysis and passive transport across membranes
free energy = RT ln([OUT]/[IN])

if negative, then transport will occur

Question 10

what is the stationary and mobile phase in chromatography

Answer 10

filter paper = stationary phase
organic solvent = mobile phase

Question 11

what is Size exclusion chromatography (SEC)

Answer 11

protein sample put through molecular exclusion gel
this gel has porous beads
meaning small molecules explore the beads before being released
whereas big ones cant get into them so they just pass straight through and are released first

Question 12

what is ion exchange chromatography

Answer 12

protein sample put through gel with -ve charged beads
> positively charged protein binds to the beads
> negatively charged protein passes striaght through

then NaCl used to ‘elute’ the protein, basically it separates into Na+ and Cl- and these ions compete with the proteins, displacing them

Question 13

How could a protein be eluted

Answer 13

eluted is just recovering the protein after seperating it from the rest of the mixture
So, could add a competitor to displace the protein from whatever it is bound to
Or change the conditions to where it loses its affinity and is released

Question 14

Affinity chromatography

Answer 14

Immobilised molecule with affinity for the protein
is used to “trap” protein of interest
Then protein is eluted

Question 15

what are the types of affinity chromatography

Answer 15

• immunoaffinity
• immobilised ligand
• lectin-based (remember, lectin binds to glycosylated proteins - cuz it binds to the sugars)
• immobilised metal affinity

Question 16

metal affinity chromatography - what protein residue binds well to tranisiton metals

Answer 16

hisidine

Question 17

hwo does metal affinity chrom work

Answer 17

metal is immobilised
protein gets engineered to add on poly histidine residues at the end terminus
then when protein passed through, the metal will bind histidine

Question 18

what part of His binds to metal proteins

Answer 18

the N in the ring without the H

Question 19

what is used to elute His bound proteins

Answer 19

Imidazole
has similar struc to His
added in excess
displaces the proteins so they can be collected

Question 20

what is IDA and NTA

Answer 20

2 acids
used to immobilise the metal
IDA = binds metal at 3 sites
NTA= binds metal at 4 sites (hence the metal being immobilised a bit stronger)

Question 21

what is special abount metal affinity chrom with 3+ metals

Answer 21

these bind phosphate groups
so can be used to seperate specifically phosphorylated versions of proteins

Question 22

what is GST-fusion affity chrom

Answer 22

• an artificially made fusion protein made
  this is the desired protein added onto a GST molecule
• GSH is immobilised in stationary phase
• GST (along with the protein attached) binds to GSH as the protein is passed through
• can be recovered with competitive elution w/ excess aqueous GST