As I mentioned in my previous blog post, this week’s post will be dedicated to explaining the process of western blotting.
First, you must understand the reason one would need to know how to do a western blot. The reason is actually quite simple. In any given tissue sample, if the concentrations of various proteins needs to be know, then a western blot is your weapon of choice. Essentially, through a several-step process, a western blot lets you see the quantities of a certain protein in a given substance.
Well, how does it work?
That’s the fun part of a western blot. We can break down the process into three steps:
1. Protein Separation
3. Protein transfer/antibody analysis
You may understand the first two steps from my previous posts, however, the third is what is interesting and unique to western blotting. When you want to examine a protein, it is easy to find via electrophoresis, but from that, you still have no idea of the quantities in which it is present.
The first part of finding out how much of a protein is present is to find the protein. This is done via the use of an antibody. Essentially, an antibody is a kind of cell engineered to neutralize another type of cell. The human body creates them in response to immune threats. Thankfully, other species do this as well, and scientists have been able to engineer these antibodies in animals. These are then harvested and used in experiments. Western blotting utilizes the antibody for the protein you want to find. You then attach a dye to the antibody and send it on its merry way to the proteins. It will then latch on the the specific protein it was engineered for, and a certain color will be displayed on the membrane upon which this process takes place.
It will then latch on the the specific protein it was engineered for, and a certain color will be displayed on the membrane upon which this process takes place. The color will be compared to a scale to see the exact quantity of the protein. It’s as easy as 1, 2, 300 dollars per micro-liter of antibody.
Hopefully that explains the process well enough for all readers.
Until next week.