My Research Position at Baldari's Lab

My visit to Tatiana Baldari's laboratories was of particular interest to me. I enjoyed the visit so much that the group joked that I currently held a position in the lab. I don't blame them. Every time Dhruv asked a question, I answered it for the researchers. Oops!

Even though I knew about the lab's procedures, I still learned a lot from the researchers. The lab works with blood samples from leukemia patients to analyze the expression of p66Shc. The tumour suppressor p53 targets p66Shc to induce apoptosis in cells in response to oxidative stress. Apoptosis is responsible for programmed cell death and it is important for controlling cell proliferation. Uncontrolled cell proliferation can lead to cancer; in this case, it can contribute to leukemia. The researchers noticed that leukemia patients lacked the p66Shc protein. To study the expression of the p66Shc, the lab performed western blots. Western blots should not be confused with northern and southern blots. Northern blot deals with RNA, southern blot analyzes DNA, and western blot reveals protein expression.

In a western blot procedure, protein is removed from cell cultures with a centrifuge. A centrifuge uses a perpendicular force to distribute the components of cells. Sodium Dodecyl Sulfate (SDS) is then added to the protein sample to give it an overall negative charge for gel electrophoresis. Electrophoresis separates the protein sample based on size. A marker is also added to the gel so that each protein's position can be compared to a standard. Then the gel in placed in a so-called “sandwich” consisting of nitrocellulose membrane, filter paper, and a red/black cover. The blots in the gel are then transferred to the nitrocellulose membrane. Primary and secondary antibodies are added onto the membrane to analyze the binding of the target protein. In this case, p66Shc antibody is added to the membrane overnight to test for the expression of the protein. During the development of the blot, if a strong band for p66Shc appears on the film, then the protein is present and expressed in the cells.

The western blot is by no means a quick and easy protocol. It takes Baldari's lab 3-4 hours to complete the procedure up to the point where results are transferred from gel to membrane. Depending on how many antibodies you want to use, reserve one day for each primary antibody.