I'm pretty excited right now because I had a sort of a mini-breakthrough. I didn't discover anything amazing or get interesting results or anything, but I solved a problem that had been bugging me for a while now.
So far in our iGEM project, I've done a half dozen western blots - none of which actually worked. I would get nice thick bands on the gels after I stained them but when I did the chemiluminescent detection, the x-ray film would come out blank without fail. I'm no stranger to this occuring because the exact same thing occured to me when I did my western for Genetics 420. I only just figured out what I've been doing wrong.
To do a western, you take a sample of your protein (either crude from freshly lysed cells or stuff you've purified) and run it through a polyacrylamide gel. This seperates out the proteins on basis of their size. From here you can do two things: you can stain the gel to visualize the bands directly on the gel or you can transfer the proteins from the gel to a nitrocellulose membrane, which can then be probed with antibodies targeted specifically against the protein that you're interested in, which have a particular enzyme attached that emits light when a certain chemical substrate is added (in short, when you expose the membrane to x-ray film, you get a dark band where your protein of interest is, keeping the background bands to zero). What I've been doing is staining the gel to get a visual idea whether or not there is actually any protein on the gel, then transfering to a membrane, probing and exposing to the x-ray film. And it has yet to work.
It seems the problem is with the order in which I've been doing this. I always stain first, then transfer. Unfortunately, this is bound to fail. The stain that we use is called Coomassie Brilliant Blue. Its a stain that stains proteins by binding to specific amino acids. One amino acid that it has a particular liking for is histidine. Therein lies the problem: all of our proteins have six histidine residues at the end - a 6xHis tag it's called. It's this tag that we use to make sure our antibodies bind to only our proteins of interest. The antibodies have been created so that they bind specifically to 6xHis tags, which do not occur naturally in E.coli1. Since I was staining first, the His tags were bound with Coomassie stain. This blocked the antibodies from binding; preventing any reaction and no bands on the x-ray film. What I should have done was transfer to the membrane first, and stained the gel after (since transfer is never 100% and some proteins remain on the gel).
I'm redoing two westerns today that didn't work before. Hopefully I will actually get results this time!
1. I have heard talk of one "mysterious" E.coli protein that seems to come up over and over when using 6xHis tags in westerns, but no one knows what it is. I dont know how true this is, but it doesnt seem to be much of a problem for researchers since His tags are probably one of the most common protein tags used in molecular biology.