QBM Exam | Questions Solved with Verified
Correct Answers | Latest Update 2024-2025
After the blocking process, what do we do with the membrane? - ANSWER We soak it in
a solution containing a primary antibody to the protein of interest.
Since the protein we are working with in a western blot is most likely denatured due to the SDS-
PAGE, what type of epitopes will the antibody target? - ANSWER Linear epitopes (primary
sequence) of 6-10 adjacent amino acids
How do we choose primary antibodies? - ANSWER 1) By whatever is available and whatever is
least expensive
2) Inject protein or epitope into animal & purify it
3) Express antigens recombinantly
4) Use antibodies specific to tag present on protein
What are some examples of tags present on proteins? (5) - ANSWER 1) GST
2) GFP
3) MBP
4) his-tag
5) FLAG-tag
What do we use to rinse off any unbound primary antibody? - ANSWER TBS-T (tris buffered sale
with tween-20)
TBS-T is a ______ used to break weak interactions. - ANSWER detergent
,What is the most frequent way to detect the presence of antibody? - ANSWER Add
secondary antibody
How do we obtain secondary antibodies? - ANSWER Inject the Fc region of an antibody into
another animal (ex/ Fc from goat to rabbit body)
Secondary antibodies can be coupled to what two enzymes to catalyze a chromogenic reaction?
- ANSWER 1) Horseradish Peroxidase (HRP)
2) Alkaline Phosphatase
How many conjugated secondary antibodies do we need to target 4 primary antibodies from
another species? - ANSWER 1
The primary/secondary system allows for multiple primary antibodies against multiple _______
to be used on a membrane. - ANSWER proteins
Is western blotting sensitive? - ANSWER Yes - extremely. It can detect picograms of a specific
protein out of thousands of proteins on a gel.
Antibodies can be detected via what four things? - ANSWER 1) Chromogenic reaction
2) Autoradiography
3) Chemiluminescence
4) Direct Infrared Fluorescence
HRP along with ____ results in a color change on the western blot. - ANSWER TMB
What does ELISA stand for? - ANSWER Enzyme-linked immunosorbent assay
, ELISA allows for the detection of what in a sample? - ANSWER Antigen or antibody
For ELISA, do we run an SDS-PAGE gel? - ANSWER No! Instead, samples are placed in small
wells and assayed. This is the difference between western blot and ELISA
Does ELISA provide information on the molecular weight of the antigen? - ANSWER No!
Does Western Blot provide information on the molecular weight of the protein bound by
the antibody? - ANSWER Yes!
For Western Blot, do we run SDS-PAGE? - ANSWER Yes
What are the two types of ELISA? - ANSWER Indirect ELISA
Sandwich ELISA
Which type of ELISA is has a setup identical to a western blot? - ANSWER Indirect ELISA
Antigen is bound to membrane, primary antibody is added to the well, followed by
secondary antibody. The solution changes color when substrate is added to the well.
Which type of ELISA is more specific and will reduce false positives, since only the antigen of
interest should remain after washing it? - ANSWER Sandwich ELISA
What is the disadvantage of the sandwich ELISA? - ANSWER It requires multiple antibodies
that recognize the target antigen which may not always be available.
What is one advantage of ELISA? - ANSWER It can read dozens of samples at once, they
are reliable, & take only minutes to perform.
The pregnancy test is an example of what type of ELISA? - ANSWER Sandwich ELISA
Correct Answers | Latest Update 2024-2025
After the blocking process, what do we do with the membrane? - ANSWER We soak it in
a solution containing a primary antibody to the protein of interest.
Since the protein we are working with in a western blot is most likely denatured due to the SDS-
PAGE, what type of epitopes will the antibody target? - ANSWER Linear epitopes (primary
sequence) of 6-10 adjacent amino acids
How do we choose primary antibodies? - ANSWER 1) By whatever is available and whatever is
least expensive
2) Inject protein or epitope into animal & purify it
3) Express antigens recombinantly
4) Use antibodies specific to tag present on protein
What are some examples of tags present on proteins? (5) - ANSWER 1) GST
2) GFP
3) MBP
4) his-tag
5) FLAG-tag
What do we use to rinse off any unbound primary antibody? - ANSWER TBS-T (tris buffered sale
with tween-20)
TBS-T is a ______ used to break weak interactions. - ANSWER detergent
,What is the most frequent way to detect the presence of antibody? - ANSWER Add
secondary antibody
How do we obtain secondary antibodies? - ANSWER Inject the Fc region of an antibody into
another animal (ex/ Fc from goat to rabbit body)
Secondary antibodies can be coupled to what two enzymes to catalyze a chromogenic reaction?
- ANSWER 1) Horseradish Peroxidase (HRP)
2) Alkaline Phosphatase
How many conjugated secondary antibodies do we need to target 4 primary antibodies from
another species? - ANSWER 1
The primary/secondary system allows for multiple primary antibodies against multiple _______
to be used on a membrane. - ANSWER proteins
Is western blotting sensitive? - ANSWER Yes - extremely. It can detect picograms of a specific
protein out of thousands of proteins on a gel.
Antibodies can be detected via what four things? - ANSWER 1) Chromogenic reaction
2) Autoradiography
3) Chemiluminescence
4) Direct Infrared Fluorescence
HRP along with ____ results in a color change on the western blot. - ANSWER TMB
What does ELISA stand for? - ANSWER Enzyme-linked immunosorbent assay
, ELISA allows for the detection of what in a sample? - ANSWER Antigen or antibody
For ELISA, do we run an SDS-PAGE gel? - ANSWER No! Instead, samples are placed in small
wells and assayed. This is the difference between western blot and ELISA
Does ELISA provide information on the molecular weight of the antigen? - ANSWER No!
Does Western Blot provide information on the molecular weight of the protein bound by
the antibody? - ANSWER Yes!
For Western Blot, do we run SDS-PAGE? - ANSWER Yes
What are the two types of ELISA? - ANSWER Indirect ELISA
Sandwich ELISA
Which type of ELISA is has a setup identical to a western blot? - ANSWER Indirect ELISA
Antigen is bound to membrane, primary antibody is added to the well, followed by
secondary antibody. The solution changes color when substrate is added to the well.
Which type of ELISA is more specific and will reduce false positives, since only the antigen of
interest should remain after washing it? - ANSWER Sandwich ELISA
What is the disadvantage of the sandwich ELISA? - ANSWER It requires multiple antibodies
that recognize the target antigen which may not always be available.
What is one advantage of ELISA? - ANSWER It can read dozens of samples at once, they
are reliable, & take only minutes to perform.
The pregnancy test is an example of what type of ELISA? - ANSWER Sandwich ELISA
