Student Research at Duke

Once again I must apologize for not being prompt with my blog posts. It seems like every day I say to myself, “I will blog today,” but then two weeks go by without anything being written. Anyway, last time I discussed how I intended to measure the activation of the protein Rac in Hela cells via cell stimulation with EGF. Well, the cells did finally grow to sufficient confluence and I made my cell lysate samples. I don’t think I have yet mentioned cell lysates, so basically they are samples made by destroying the cells but retaining all of the proteins and components in the cell. This experiment was run by doing what is called a Rac pulldown assay, in which the cell lysates are mixed with a substance called GST-PBD. GST-PBD selectively binds to activated Rac, so in this way we are able to isolate the active Rac and run the samples through a western blot (this I know I have mentioned!) to measure the activation.

Unfortunately, all the time spent making the lysate samples and running western blots proved to be fruitless, as the phosphor imaging scanner showed dirty protein membranes with absolutely no Rac bands. I have yet to determine what error caused this, as the scanner is in fact functioning correctly. This was a most frustrating occurrence, as it seemed that several days of work went down the drain.

After the unpleasantness, Hideji and I regrouped and decided to perform a simpler version of that experiment using 3T3 cells, which are fibroblast cells from mice. In some ways I am glad that I had to redo some of this experiment because I am finding that each time I make the lysates, along with doing the western blot, my technique gets a little better. This time the scans came out much better and I will be obtaining data next week by quantifying the Rac bands from the scans. Although I still have to “officially” analyze the scans, they look promising and I am very glad to end the week in this way.

Once again thank you for reading and although there are only two weeks left in the program I will give it my best effort to update my blog reguarly (well, maybe semi-regularly!).