lecture 6 - assessing purity with SDS-PAGE

Question 1

step 1 to assess purity and yeild

Answer 1

• measure target protien

- measure total protein

Question 2

how do we measure purity?

Answer 2

amount of target protein/total protein

Question 3

how do we measure yeild?

Answer 3

amount of target

Question 4

measurments can be:

Answer 4

semi-quantitative and quantitative

Question 5

SDS-PAGE

Answer 5

• sodium dodecyl sulphate polysacrylamise gel electrophoresis
• most common semi-quantitative measurement

Question 6

what is SDS

Answer 6

SDS = nionic detergent
-unfolds proteins & coats them w uniform charge/mass ratio
~1 SDS/ amino acid residue
-separates oligomers into their constituent subunits or momomers

Question 7

acceleration in SDS-PAGE

Answer 7

• F=ma, a=F/m
• F and m are both proportional to MW
• acceleration in an electric feild is the same for all proteins BUT proteins are slowed by the gel (cross linked polymer)

Question 8

speed through SDS-PAGE gel is determined by?

Answer 8

size

• larger = slower
• smaller = faster

Question 9

how does SDS-PAGE work?

Answer 9

• different samples are run through a gel
• gel has electric feild going from negative to postitve
• smaller proteins travel further down the gel & larger ones stay towards the top

Question 10

how are proteins on SDS-PAGE visualized?

Answer 10

by staining

• e.g. coomassie blue binds to hydrophobic regions of protein
• amount of die in a band is proportional to the mass of the protein (MW x moles)

Question 11

example question using SDS-PAGE:

equimolar amounts of 2 proteins (90 kDa:45 kDa) are run through the gel in a 1:4 ratio. The 45k Da protein shows:

(a) 4x as much stain as the 90 kDa protein
(b) 2x as much stain as the 90 kDa protein
(c) half the stain of the 90 kDa protein
(d) the same amount of stain as the 90 kDa protein

Answer 11

answer: (b)

1x90 = 90
4x45= 180 

180/90= 2

Question 12

why is SDS-PAGE useful

Answer 12

• can estimate relative amounts of protein

- can estimate stoichiometry of oligomeric proteins

Question 13

how to estimate MW using SDS-PAGE

Answer 13

• compare position of bonds to MW standards

- use graph to compare how far travelled (position in x axis) to MW on y axis

Question 14

list a potential complication for using SDS-PAGE to estimate MW

Answer 14

• cis residues can form a disulpide bond (covalent linkage) by oxidation
• found in some extracellular proteins
• if not reduced:
• protein may not unfold completely (appears smaller)
• multimeric protiens may have inter-subunit disalphides (appears larger)

Question 15

how do we tell if the potential complication occured?

Answer 15

• SDS-PAGE normally run with a reducing agent

- if run same protein with an oxidizing agent and result is different…

Question 16

example question: complication

protein run on a reducing gel showed 40 kDa
same protein run on oxidizing agent showed 80 kDa
what’s going on

Answer 16

implies homodimer of 40 kDa subunits linked by disalphides