Week 8

8/6
At the start of my final week, I begin by taking pictures of the cover slides that I made. We took pictures of cells with F-Actin staining—this was to make sure that the shEGFP did not affect the cytoskeleton in any way and allowed them to be used as a control. I split down more of my cells and replaced the media in all of them. I also plated 3mL of EGFP expressing cells with shEGFP and Clone A cells with shEGFP in a 6-well plate. I then extracted and purified RNA and protein lysates from 3.2 virus cells, Clone A cells, and Clone A shEGFP cells the same way I did the previous week.

8/7
Bad news today. I put the RNA through PCR and later, when they came out, I checked them under the UVP machine for any β4 knockdown. Unfortunately, the β4 did not appear knocked down at the RNA level, so my only hope was for the cell lysates to show a β4 knockdown. I began to develop the lysates, running them through an 8% gel and then coating them with 505 antibody overnight. Before I left, I coated 12 wells of a 24 well plate with laminin for a migration assay.

8/8
Right when I came in today I began developing my Western Blot. I washed off the antibody and then applied a secondary anti-rabbit antibody. Once this was washed off, I applied the luminescent developing material and then took the membrane to the dark room. Waiting the 5 minutes for the film to come out of the machine seemed like the longest 5 minutes of my life. The film finally rolled out of the developing machine and I could see that all my hard work had finally paid off. The β4 levels in the protein of the cells containing my virus have dropped dramatically.

Since my experiment finally received some good data, we began to hurry on the migration assay because my presentation is tomorrow and I need some conclusions. I spent hours waiting for my migration plate to finish incubating after I had coated the wells with special media and added my cells, then I anxiously washed the cells and applied the luminescent developing chemicals. We hurried to the microscope and saw that the β4 migration was only slightly reduced, giving me inconclusive results. Still, my experiment proved a success, even without concrete results. I had successfully knocked down the β4 levels in Clone A cells, which is all that mattered.

8/9
Today is my last day. I gave my PowerPoint presentation to the lab, which I will also present at Brooks in the fall. After going over my data and results, I cleaned up my area and we said our goodbyes. Thus concludes my experience at UMASS Medical Center.