Question

Question 1. You are a cancer researcher studying skin cancer cells that have become metastatic (ie. they've migrated from the site of the original tumor to establish new secondary tumors). You analyze skin samples from an unaffected patient and from one with the metastatic cancer. After a series of detergent solubilization and centrifugation steps, you separate pellet samples from each tissue type by gel electrophoresis and find the following results: skin normal cancer tissue tissue 250 Da 120kDa 75kDa- 60kDa 30kDa- A. What is the difference between the normal skin sample versus the skin cancer tissue sample? (2 points) B. Which protein would you select for further study? (2 points) C. Many proteins work in complexes with other proteins. How could you determine if the cancer protein in question above has one or more intracellular binding partners? (6 points)

Answer 1

A. in normal skin sample all the proteins are generally expressed, means neither of them are either over expressed or under expressed which signifies that the cells does not have cancer. In skin cancer tissue the protein B is over expressed which implies that the gene encoding the protein B has been affected due to cancer.

B. I will select protein B for further studies as this protein is over expressed in the cancerous tissue.

C. generally all the proteins work in a combined manner, means they interact among themselves and carry out a function. in this case also the cancer protein might have some interactions with the other proteins. the interactions can be studied using various techniques, such as:

a) co-IP - it stands for co immunoprecipitation. in this method the target protein binds to the interacting protein which is bound to the antigen and the antigen is bound to the antibody. this helps in precipitating both the proteins from the lysate.

b) pull down assay- this is similar to the co-IP but the difference here is that instead of using antibodies we use a pre-prepared protein(known as bait) to purify any protein which binds to the bait protein.

c) cross-linking protein interaction- in this method the proteins are cross linked using various cross linking agents and then the interaction is seen in western blotting.

d) label transfer protein interaction- in this method the interacting molecules are cross linked using a labelled cross linking agent. the bait protein is first cross linked to labelled cross linking agent and then the complex is linked to prey protein. after this the linkage is broken from the bait protein and the prey protein remains attached to the cross linking agent.