The Highs are worth the Lows

My first Western Blot ever. I am thinking of framing it. I was so proud of myself. I did everything from scratch. I know it may seem simple and everyone in the biology world might know how to do it, but it was my first and it was a big thing for me. I casted the stacking and the resolving gel. Again I have learned the WHY of everything. I now know the importance of the pH difference between the stalking and the resolving gel and the importance of the amount of polyacrylamide one adds (depends on the size of the protein of interest). If you study a big protein then you add less polyacrylamide in order for the big proteins to run faster down the gel. The Ammonia Persulphate Solution (APS) added to preparation of gel acts as an initiator and the TEMED as a catalyst so you add those two components last in order to begin polymerization of your gel (not before or it becomes a solid before you can cast it). I heated my samples with loading buffer which contained SDS and loaded it with marker and previously collected samples. I covered everything with running buffer at working conditions (1x this term is also new to me). Then transferred my proteins from the gel to a membrane via current again. Once my proteins were in the membrane, I blocked the membrane with milk, yeah milk, inorder to prevent unspecific binding of the primary antibody and then added the diluted rabbit antibody overnight which was specific to protein of interest which was CHIP in this case. You have to pay attention to where your proteins are at all times and having a marker with protein sizes helps. CHIP was around 35kDa. Then I had to come in the next morning to do washes and place secondary antibody which was anti-rabbit. This secondary antibody is special in that it has Horse Radish Peroxidase. It is used to locate the protein of interest since it is an enzyme that provides a detectable signal. In order to obtain this signal you have to go down to the dark room just like photography to develop film. In the dark room you provide the enzyme with substrate and then it turns it into product that can be detected with the film. It is the coolest thing. I have also used another technique where you can scan the membrane and it detects fluorescence but here you have to use a different secondary antibody, I do not know the specifics just yet, but it’s easily done which I like aswell.
I am telling you all this not because I want to bore you, but to show you how much I have learned… I do not want to simply to tell you that I have learned a lot… I want you to believe me! The more I think about it, the more grateful I am for this experience. It was extremely hands on. It showed me to think and to practice. Yeah it was a lot of mistakes. So many, but that’s what really makes me appreciate it. I am so happy. This has totally been worth it, every minute.