Reference no: EM133191266

SDS-PAGE and western blotting assignment

Question 1) Define SDS-PAGE and mention one other use for this technique other than western blotting

Question 2) During SDS polyacrylamide gel electrophoresis (SDS-PAGE):

a. Why is it necessary to add SDS to both the loading buffer as well as the running buffer?

b. What would happen to the protein banding pattern if SDS was not added to the gel and buffer?

Question 3) Explain what the primary and secondary antibodies recognise and how this leads to band detection

Question 4) Why is it necessary to transfer proteins from a gel to nitrocellulose or PVDF membrane during western blotting?

Question 5) Your research supervisor suggests to you that after obtaining your protein samples you would need to perform a BCA assay.

a. What is the principle of the BCA assay and what information does it provide?
b. Why would a researcher need to perform an assay like the BCA assay before running a western blot or ELISA?

Question 6) You and a fellow researcher are both performing SDS-PAGE however you notice that your colleague places the gel cassette incorrectly (opposite orientation - anode to cathode instead of cathode to anode) between the anode and cathode.
a. What effect would this have on the samples ability to migrate through the gel?
b. Why would the effect mentioned in (a.) occur?

Question 7) A researcher performs ponceau staining on their membrane where bands ran and separated fine except that he/she notices a few areas on the membrane that have distorted circular shapes that did not stain.

What could have caused this and how can the researcher ensure that it does not happen in the future?

Question 8) A researcher decides to carry out a western blot however makes a number of mistakes during the western blotting procedure.

a. When performing band detection, he notices that around 8-10 bands are detected with high background in each lane and that dark smears appear in the corners of the membrane. Provide reasons why this occurred.

b. Troubleshoot possible steps the researcher could take to prevent this issue in future.

9) A researcher casts an SDS-PAGE gel but forgets to overlay the gel with SDS or ethanol. What effects could this potentially have on the experimental procedure?

Question 10) The following images of gel stains, membrane stain and western blotting visualisation all indicate various possible problems that went wrong during the experimental procedure.

The below gel stain and ponceau stain both depict the exact same problem with the samples. What is it, and how would you rectify it?

The ponceau stain and western blot below both depict the exact same problem with the samples. What is it, and how would you rectify it?

The issues present in the below western blot depict an issue that occurred after transfer. Suggest 3 possible reasons that could have caused this?

Attachment:- western blotting assignment.rar