Western Blot Flashcards
What is Western blotting used for?
To detect the presence of a specific protein in a protein mixture extracted from cells
To evaluate the size of a protein
Measure the amount of protein present
What are the three steps in western blotting?
Separation by size
Protein transfer to solid support
Detection of target protein
How are antibodies for Western blot made?
Antigen injected into animal and its immune system produces a response
What do polyclonal antibodies consist of?
Mixed pool of Ig molecules
These bind to different epitopes found on a single antigen
What do monoclonal antibodies bind to?
A single epitope on the target antigen
Which antibody type has a higher probability of detecting the protein?
Polyclonal
Bc binds to various segments
Which antibody type is more sensitive?
Polyclonal
Bc amplification occurs are every binding site
What can cell lysates be prepared from?
Transfected cells carrying a protein expression vector
OR
immortalised cell lines known to express the target protein
At which temp should cell lysates be prepared?
Cold/on ice
Why may sonication be required?
To fully release the protein from certain cell and tissue samples
What is present in the gel of most western blots?
SDS
Reducing agent
Why does SDS-PAGE work?
The proteins will be negatively charged due to the SDS
They then migrate in an electric field through the gel to the positive electrode
The mass:charge ratio gets normalised by the binding to SDS along the gel
Bigger proteins then go further
How are the proteins transferred onto a blotting membrane?
An electric field is run through perpendicular to the angle of the gel
What can be added to the gel running buffer to help the proteins bind to the blot?
Methanol
What are the advantages of using a nitrocellulose gel?
Excellent protein binding and retention capabilities
What are the disadvantages of nitrocellulose gel?
Brittle and less effective if re-use needed
Why are gloves always worn in Western blot?
Oils in hands can interfere with the signal on the blotting membrane
Why only one band visible in Western blot film?
Antibodies should only bind to that protein
The unbound is then washed off
Which gel is acidic and which is alkaline?
Higher, stacking gel - slightly acidic
Lower, resolving gel - basic
Which is more reliable, semi-dry or wet?
Wet
What may explain unusual or unexpected bands in the results?
Protease degradation
What may explain blurry bands in the results?
High voltage or air bubbles in transfer
Why may no bands appear?
Improper antibody
Low concentration of antigen
Prolonged washing
Contamination
What may lead to patchy/uneven spots on the blot?
Air bubbles
Which neurotoxin do the PAGE gels contains?
Acrylamide
Through which type of bonding do proteins bind to nitrocellulose?
Hydrophobic interaction
What are the 3 components in stage 3 of western blotting?
Blocking
Antibody incubation
Detection
What is the “blocking” component of protein detection?
Incubation of the blot with a protein or detergent
To prevent nonspecific binding of the antibodies to the membrane
What is used as a blocking agent in Western blot?
Non fat dried milk
Describe the antibody incubation of protein detection
Labelled primary antibody introduced to the blot
This binds to the protein on the nitrocellulose membrane
What primary antibodies can be used as “house-keeping” or control?
anti-GAPDH
Actin
Describe the final part of protein detection
Secondary antibody introduced which binds to the primary
This oxidises luminol to ground state in the presence of H2O2 in the enhanced chemiluminescence (ECL) to give off visible light when developed
How is the molecular weight of the protein quantified in WB?
Compared to other molecular weight markers on the side of the blot
Or
Densitometer can be used to scan the blot of film and use software to compare the signal bands
What is the purpose of the housekeeping protein?
To convey that an equal amount of protein lysate was loaded onto the gels for the mock and the DNA
If bands are equal size/intensity = equal amount of protein
Are smaller proteins positively or negatively charged?
Negative
The higher the gel & percentage the higher/lower the size of the protein?
Lower, in general
Describe how the transfer stack is arranged?
Neg electrode Sponge Filter paper Gel Membrane Filter paper Sponge Pos electrode
What does electrophoresis let you separate proteins by?
Weight (kDa)
Why were the samples lysed with BMCE and SDS?
Denature proteins to be linear
Why was the protein sample put on a heat block?
Aid denaturation
What is the purpose of the Ponceau S?
To help visualise the bands if they are there
What kind of molecules is non-fat dried milk not good for?
Fluorescent antibodies
What can be used instead of 5% Marvel as a blocking agent?
BSA
Why was the secondary antibody an anti-rabbit antibody?
Because the primary was made in rabbit
