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Western Blotting Assignment post lab

Woriax and Santisteban, Spring 2021
Spring 2021
ASSIGNMENT FOR Western Blot of Various Proteins Lab
1. What is the purpose of the 1◦ antibody versus the 2◦ antibody?
The primary antibody directly attaches to the protein of interest. The secondary antibody
is used in detection so it will bind to the primary antibody and allow us to visually see it.
Without the secondary antibody you would not necessarily be able to detect the protien
with just the primary antibody.
2. Suppose you used a 1◦ antibody that was raised in mouse and then used a 2◦ antibody that
was anti-chicken. What would be the outcome of your experiment and why?
The secondary antibody would not be able to bind with the primary antibody. This would
mean you would not be able to detect the primary antibody or the protein it has attached
to. In our experiment we used a primary protein made in a rabbit and then used an antirabbit antibody made from goat as our secondary antibody.
3. What is the difference between a CDR vs epitope?
An epitope is the area of an antigen molecule that the antibody attaches itself to. CDR
(complimentary- determining regions) on the other hand is the area of the antibody that
binds to the antigen.
4. What possible problems might you anticipate if you erroneously skipped the blocking step?
If you were to skip the blocking step than the antibodies would bind to the membrane
nonspecifically because you did not block the remaining sites.
5. What information is provided by staining the blot with Ponceau S or Fast Green?
- Allows us to visualize all proteins that are present on the blot.
6. How would you prepare 50 mL of a 1:3000 dilution of antibody?
_____16.67 microliters__(vol) antibody to _____50mL____ (vol) TBST.