SDS-PAGE and WB Flashcards
(28 cards)
What is an SDS-PAGE used to estimate?
- the size of a protein compared to MW standards
- the amount in a mixture (thickness of the band)
- the purity (number of the bands)
What is the function of SDS and 2-ME in an SDS-PAGE?
SDS: covers proteins in a net negative charge –> linerarizes the proteins (denatures)
2-ME: reduces disulphide bonds (denatures)
What affects the range of separation of proteins in an SDS-PAGE?
the percentage of polyacrylamide used
What is the purpose of the stacking gel?
has looser pores to allow proteins to line up
Why should the running gel be overlayed with ethanol or methanol?
gives a flat surface and since the polymerization of acrylamide is inhibited by oxygen, this will speed up polymerization
What is the minimum protein loading per well for Coomasie blue and silver staining?
CB = 0.1ug
S = 2ng
What is the maximum protein loading per well?
40ug
How does KCl affect SDS? What can be done about samples containing KCl?
KCl causes SDS to precipitate
if your sample contains KCl, you should dilute them or methanol precipitate them and resuspend in sample buffer
If your sample buffer is yellow, what does this mean?
it’s at the wrong pH
What should you do if you have leftover unpolymerized acyrlamide? Why?
polymerize the rest with excess catalyst and dispose of with solid waste, this must be done because acrylamide is a neurtoxin
How long is ammonium persulfate and TEMED good for? can they be stored?
ammonium persulfate should be made up fresh
TEMED should be stored in the refrigerator and its good for about a year
When should you use silver staining over coomassie blue?
silver staining is more sensitive than CB, so if you cannot see your bands after staining with CB, use silver
What is a native PAGE? Why would you use it?
What = run a PAGE under non-denaturing conditions –> leave out SDS and 2-ME
Why = can identify and analyze multi-protein complexes and dimerizaiton
What things should you consider with your sample prep for an SDS-PAGE?
- is the target protein soluble/cytoplasmic, insoluble or membrane-bound?
- are additional buffer components (e.g. detergents, enzymatic inhibitors) required for solubilization, fractionation or maintenance of post-translational modifications
- what is the method of protein quantification, will buffer components interfere?
What things should you consider with your PAGE?
- what concentration is most appropriate?
- what running buffer is most suitable?
- how long and what voltage to run the tank?
What can a western blot (WB) do that a protein gel cannot?
identifies a specific protein
What should you consider with your electro-transfer for a WB?
- what is the most suitable membrane material and pore size?
- is methanol excluded in the buffer?
- should membrane staining occur to assess transfer efficiency?
What should you consider with your blocking for a WB?
- which blocking reagent is most suitable?
- what concentration and what buffer should be used?
What are the two major factors that affect the efficiency of a WB?
- the elution from the gel: use the lowest % acrylamide that will allow resolution + higher MW proteins blot poorly
- efficiency of binding to the membrane: nitrocellulose non-covalently binds proteins
Describe the proper order of layers for a semi-dry transfer
cathode - anode: two buffer soaked filter papers - gel - membrane - two buffer soaked filter papers
Describe the proper order of layers for a wet transfer
cathode - anode: support grid - pads - filter paper - gel - membrane - filter paper - pads - support grid
What should you consider for the primary Ab in a WB?
- what are the general characteristics of the primary Ab?
- is it specific towards native or denatured proteins and is the eptiope sequence/region known?
- are additional bands known/present (degradation products or isoforms)?
- have appropriate controls been run to determine specificity?
- is the Ab. specific to a post-translational modification?
What should you consider for your secondary Ab in a WB?
- what is the label conjugation
- is the secondary Ab specific to the primary isotype?
- is there detectable/over-exposed signal, if so is an Ab dilution curve required?
What should you consider for your detection in a WB?
- what is the label conjugation (e.g. HRP or fluorescence)?
- is fluorescence multiplexing suitable?
- have the Abs been stored correctly?
- can the membrane be successfully stripped and reprobed?
