SDS-PAGE and WB

Question 1

What is an SDS-PAGE used to estimate?

Answer 1

1. the size of a protein compared to MW standards
2. the amount in a mixture (thickness of the band)
3. the purity (number of the bands)

Question 2

What is the function of SDS and 2-ME in an SDS-PAGE?

Answer 2

SDS: covers proteins in a net negative charge –> linerarizes the proteins (denatures)

2-ME: reduces disulphide bonds (denatures)

Question 3

What affects the range of separation of proteins in an SDS-PAGE?

Answer 3

the percentage of polyacrylamide used

Question 4

What is the purpose of the stacking gel?

Answer 4

has looser pores to allow proteins to line up

Question 5

Why should the running gel be overlayed with ethanol or methanol?

Answer 5

gives a flat surface and since the polymerization of acrylamide is inhibited by oxygen, this will speed up polymerization

Question 6

What is the minimum protein loading per well for Coomasie blue and silver staining?

Answer 6

CB = 0.1ug
S = 2ng

Question 7

What is the maximum protein loading per well?

Answer 7

40ug

Question 8

How does KCl affect SDS? What can be done about samples containing KCl?

Answer 8

KCl causes SDS to precipitate

if your sample contains KCl, you should dilute them or methanol precipitate them and resuspend in sample buffer

Question 9

If your sample buffer is yellow, what does this mean?

Answer 9

it’s at the wrong pH

Question 10

What should you do if you have leftover unpolymerized acyrlamide? Why?

Answer 10

polymerize the rest with excess catalyst and dispose of with solid waste, this must be done because acrylamide is a neurtoxin

Question 11

How long is ammonium persulfate and TEMED good for? can they be stored?

Answer 11

ammonium persulfate should be made up fresh

TEMED should be stored in the refrigerator and its good for about a year

Question 12

When should you use silver staining over coomassie blue?

Answer 12

silver staining is more sensitive than CB, so if you cannot see your bands after staining with CB, use silver

Question 13

What is a native PAGE? Why would you use it?

Answer 13

What = run a PAGE under non-denaturing conditions –> leave out SDS and 2-ME

Why = can identify and analyze multi-protein complexes and dimerizaiton

Question 14

What things should you consider with your sample prep for an SDS-PAGE?

Answer 14

1. is the target protein soluble/cytoplasmic, insoluble or membrane-bound?
2. are additional buffer components (e.g. detergents, enzymatic inhibitors) required for solubilization, fractionation or maintenance of post-translational modifications
3. what is the method of protein quantification, will buffer components interfere?

Question 15

What things should you consider with your PAGE?

Answer 15

1. what concentration is most appropriate?
2. what running buffer is most suitable?
3. how long and what voltage to run the tank?

Question 16

What can a western blot (WB) do that a protein gel cannot?

Answer 16

identifies a specific protein

Question 17

What should you consider with your electro-transfer for a WB?

Answer 17

1. what is the most suitable membrane material and pore size?
2. is methanol excluded in the buffer?
3. should membrane staining occur to assess transfer efficiency?

Question 18

What should you consider with your blocking for a WB?

Answer 18

1. which blocking reagent is most suitable?
2. what concentration and what buffer should be used?

Question 19

What are the two major factors that affect the efficiency of a WB?

Answer 19

1. the elution from the gel: use the lowest % acrylamide that will allow resolution + higher MW proteins blot poorly
2. efficiency of binding to the membrane: nitrocellulose non-covalently binds proteins

Question 20

Describe the proper order of layers for a semi-dry transfer

Answer 20

cathode - anode: two buffer soaked filter papers - gel - membrane - two buffer soaked filter papers

Question 21

Describe the proper order of layers for a wet transfer

Answer 21

cathode - anode: support grid - pads - filter paper - gel - membrane - filter paper - pads - support grid

Question 22

What should you consider for the primary Ab in a WB?

Answer 22

1. what are the general characteristics of the primary Ab?
2. is it specific towards native or denatured proteins and is the eptiope sequence/region known?
3. are additional bands known/present (degradation products or isoforms)?
4. have appropriate controls been run to determine specificity?
5. is the Ab. specific to a post-translational modification?

Question 23

What should you consider for your secondary Ab in a WB?

Answer 23

1. what is the label conjugation
2. is the secondary Ab specific to the primary isotype?
3. is there detectable/over-exposed signal, if so is an Ab dilution curve required?

Question 24

What should you consider for your detection in a WB?

Answer 24

1. what is the label conjugation (e.g. HRP or fluorescence)?
2. is fluorescence multiplexing suitable?
3. have the Abs been stored correctly?
4. can the membrane be successfully stripped and reprobed?

Question 25

What is the most sensitive detection method?

Answer 25

radio-labelling (125 I of staph protein A or strep protein G)

Question 26

How does a far western work? Why would you use it?

Answer 26

separate prey proteins by gel electrophoresis –> probe membrane/gel with a labeled bait protein –> prey and bait protein interact –> add substrate to develop

use = detection of protein-protein interactions

Question 27

What is the purpose of a southwestern blot?

Answer 27

look for DNA binding proteins

Question 28

How does co-immunoprecipitation work? Why would you use it?

Answer 28

add Ab that is specific to one protein that’s in a protein complex –> immunoprecipitates the entire protein complex for a protein mixture