Week 4 – Western Blot

Western Blot — No it’s not a type of Western painting featuring Clint Eastwood. It’s something much more important to my project.

In my 2nd post, I explained that the 2nd part of my project features Western Blot. Western Blot is used to monitor protein expression of specific genes. In this case, I monitor the expression of the p53, p21, NOXA, PUMA, and SOD2 genes. p53 and p21 are tumor suppressor genes. NOXA and PUMA are genes that initiate apoptosis, and SOD2 is a gene that shows cell stress (as in how toxic the drugs are to the cells). I know if the treatment has successfully entered the cells if all of these proteins are expressed. If not, I know something went wrong in the experimental procedure.

The basic procedure for Western Blot is: Extract the proteins; denature all non-proteins by making a master-mix of DNAases, RNA-ases, lipidases, and protease inhibitors; Add loading dye; run through the gel; transfer the bands to a membrane; image the membrane. The reason we must image the membrane is to calculate the ROI (Rate of intensity) values, which tell me exactly how much of the proteins are being expressed. I will show how I analyze the ROI data in the next post. Nevertheless, in the picture below, you will see a compilation of all my western blot data from 2 trials. Overall, the results are precise, with no drastic differences in expression. The proteins are being expressed as they should, and overall the Dox 1.25 and Dox 1.25+Pac conditions show the most promising results.

I do this every trial, and it may be a pain since it takes about 10-12 hours, but it’s definitely worth it…right?