MCB 253 Final Exam Questions With Verified Answers
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MCB 253 Final Exam Questions With
Verified Answers
Why is a native gel called a native gel? - answera native gel is a gel that allows for proteins
to be run in their native (natural folded) state. native gels dont use buffer to break down proteins
what is a native gel used to obtain? - answerth...
MCB 253 Final Exam Questions With
Verified Answers
Why is a native gel called a native gel? - answer✔✔a native gel is a gel that allows for proteins
to be run in their native (natural folded) state. native gels dont use buffer to break down proteins
what is a native gel used to obtain? - answer✔✔the charges of a protein as well as its isoelectric
points
what type of molecules don't do well in a native gel? - answer✔✔molecules with proteins that
widely differ in size.
What is SDS-PAGE used for? - answer✔✔used to determine the molecular weight of proteins as
well as the relative abundance of major proteins (# of polypeptides)
How does SDS buffer work? - answer✔✔-SDS buffer denatures the hydrophobic interactions of
proteins and gives them an overall negative charge bc SDS is an anionic detergent
-thus proteins have the same charge to mass ratio, meaning that the bigger the protein, the more
negative it is.
-therefore proteins migrate in the gel only on the basis of mass
How does SDS-PAGE work? - answer✔✔after the anionic detergent (sds buffer) attached to the
proteins to give them a net negative charge, the gel is run and the proteins will move vertically
from the (-) cathode to the (+) anode. As the proteins move through the acrylamide gel, the larger
molecules get stuck at the top while the smaller ones are able to move farther along. After the gel
has finished running, you can measure the distances traveled and create a semi-log graph of dist
migrated as a function of molecular weight.
What is the isoelectric point? - answer✔✔The pH at which there is no net charge on a molecule
What determines the migration rate of proteins through the gels? - answer✔✔Size. smaller can
weave through the dense acrylamide with greater ease.
What is the purpose of the MW marker ladder? - answer✔✔allows for you to compare the bands
of the unknown. you make a semi log graph to find the distances that dont perfectly line up with
the standard.
what is the purpose of the western blot? - answer✔✔In utilizing the western blot procedure we
can determine the identity of our protein. After a normal SDS page with coomassie blue, the
samples can no longer be used, but with western blot the researcher is able to transfer the
proteins to a membrane.
Sum up the western blot - answer✔✔Run gel, transfer gel contents to nitrocellulose membrane
via a blotting sandwich (sponge, filter paper, gel, membrane, filter paper, sponge) and then
running the gel. next the membrane was treated with blocking buffer to prevent non specific
binding. then it was treated with primary antibodies, washed with washing buffer, treated with
secondary antibodies, washed again, and finally treated with HRP color development (hydrogen
peroxide) to allow for visualization.
explain the difference in visualization methods of SDS PAGE and western blot - answer✔✔SDS
PAGE uses coomassie blue which prevents further use of the protein.
western blot - HRP color development which binds to the secondary AB following AB
treatment.
What do you expect to see on a protein gel after it is run? How about on a WB membrane after it
is developed? - answer✔✔-After running the protein gel, you would expect to visualize bands
next to the standard
-You would see a band on the membrane containing the primary antibody for the protein of
interest (anti-protein)
how can you confirm that your protein sample is in fact the specific protein with a native gel? -
answer✔✔the charge and isoelectric point will be the same
how can you confirm that your protein sample is in fact the specific protein with SDS PAGE? -
answer✔✔molecular weight is same
how can you confirm that your protein sample is in fact the specific protein with a western blot?
- answer✔✔the same antibody will bind to the epitope
monoclonal antibodies - answer✔✔Antibodies produced by a single clone of B lymphocytes and
that are therefore identical in structure and antigen specificity and thus can bind to a single
epitope.
polyclonal antibodies - answer✔✔Antibodies produced by injecting animals with a specific
antigen. A series of antibodies are produced responding to a variety of different sites on the
antigen. Therefore can bind to multiple epitopes.
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