|GIANTMicrobe of antibody (Y shape) and antigen (red). |
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The first step in any Western is to obtain the protein from your experimental sample and then separate it out on a gel. Then you place the gel and a special filter membrane into a sandwich of filter paper and sponges. The sandwich is put into an electrical current, the gel towards the negative pole and the membrane towards the positive. This electrical current moves the protein from the gel into the membrane. The membranes act as protein fly paper, trapping them while allowing smaller molecules to pass through small pores.
|building a sandwich|
Once sufficiently blocked (and this takes hours), the primary antibody is added and allowed to incubate for even more hours. Then a few washes are performed to remove any excess antibody from the membrane. Next comes the secondary antibody step. Then even more washing. Once this is completed, the blot can be developed. We use a chemiluminescent and a special camera to capture the bands. The only areas of the membrane that will glow are those where the primary antibody has attached to the protein and the secondary antibody has attached to the primary antibody.
|One of my early PhD Westerns, it's not that pretty.. I will do better!|
Now back in my previous grad school life, I was using a more specific antibody that only detected one band. This was pre-chemiluminescent. We had a developing buffer that had to be continually monitored so that the membrane could be quickly moved out of the developing buffer into water or else the background would interfere. As you can see in the blot below, some of the bands disappeared, meaning the protein was decreased in the environmental conditions.
|One of my M.S. blots|
The power of Western blots should not be underestimated. It takes a few days, but it is well worth the effort as it provides a clear picture of how the protein levels are, or are not, changing during your experiment.