WorksheetsWestern Blot Review
Total questions: 15
Worksheet time: 8mins
After proteins are run on an SDS-PAGE gel, a transfer is executed. What is the purpose of the transfer in Western blot protocol?
Denature the proteins in the sample
Visualize the proteins run on the gel
Move proteins from the SDS-PAGE gel to a nitrocellulose membrane
Probe the gel with an antibody to detect a protein of interest
Why is it called immunoblott?
because it involves human antibody
because the membrane contains antibody
because it applies fluorescent antibodies
because it applies antibody to tag protein
because the protein of interest is an antibody
What is the purpose of washing in every step in western blotting?
to ensure a stable pH environment in the tank
to avoid protein degradation
to remove any unbound antibodies
to moisten the transfer membrane
to remove any contaminating residues
State the purpose of blocking
to block nonspecific antibody from binding to protein
to avoid non specific binding of antibody
to avoid antibodies from binding to protein
to prevent protein from being washed off
to prevent unwanted contamination towards proteins
Primary antibody targets..
nitrocellulose surface
specific protein
primary antibody
conjugated secondary antibody
fluorochrome
Conjugated secondary antibody targets..
nitrocellulose surface
specific protein
primary antibody
conjugated secondary antibody
fluorochrome
Which of the following probes are most commonly used in Western blotting?
Nucleic acids
Bovine serum albumin
Antibodies
Nonfat dry milk
True or false: In western blotting proteins are detected through autoradiography methods with non-fluorescent compounds
True
False
Why are buffers used in Electrophoresis?
Conduct electricity
Remove salts
Add extension energy
Dissolve excess DNA
How do you know you used the wrong membrane?
the ponceau s stain isn't sticking to the membrane
the membrane glows bright blow in the image
only the bands are glowing, nothing else
True or False: The best way to normalize western blot data is to use a housekeeping protein.
true
false
The biggest thing to remember when running the gel is (according to Luay):
Make sure you load the samples and ladder in a way that looks nice and is easy to understand.
Remove the green tape!!
Use the right size lid for the right size gel box
What is the order of the western blot sandwich?
Sponge
Membrane
Gel
Sponge
Membrane
Gel
Sponge
Sponge
Sponge
Gel
Membrane
Sponge
When is the best time to get lunch (assume you take an hour)?
While the camera is taking pictures of the membrane
Waiting for membrane washing to finish
Waiting for the gel to run until the ladder touches the bottom
What is running buffer made of?
SDS + glycerol
Glucose + SDS
PBS + TriHCl + H20
