I started working on my first Western blot last Monday and started a new set of samples this week. It is probably the most difficult thing I’ve had to implement in my research. It’s just so easy to do one thing wrong and mess the entire thing up. Today, I left my lab after 7 pm, but none of that matters because of the four proteins I’m testing for, only one gave a result. So I’ll be starting the entire process over.
For those who don’t about protein extraction and blotting, Western blot is a technique used to essentially indicate whether or not an expected protein is present in a sample, similar to how PCR and gel electrophoresis indicate whether an expected gene (or set of genes) is present.
The first step to Western blot is to make an agarose gel. This is a lomger and more complicated process than making the agarose gel for electrophoresis because these are smaller, more sensitive, and made of two separate layers, which must be created one at a time. Once the gel is finished, it must not have bubbles in it that would make the blot unreadable.
Proteins must be extracted from whole leaves by grinding the samples. Then, protein extract is combined with buffers and loaded into wells of an acceptable gel. This runs fro a few hours essentially by electrophoresis. Then proteins are transferred to membranes which are put through about 100 different rinses before they can be analyzed. The entire process takes days and only maybe works. It’s great. I love it.