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10 months ago
Thursday, November 13, 2008
Protein suggestions anyone??
Okay so the western blot from hell did not work, as expected. I am not sure if it did not work b/c the antibody whose specificity I was trying to confirm did not work or b/c there was not enough protein on the blot or for some other random methodological error. I rerunning the gel and will stain for my protein of interest, along with another abundant fly protein that does not run at the same size of my protein. I am having problems finding that well known fly protein. My protein should run somewhere between 50 kDa and 30 kDa. Actin is out, Tubulin is out, spectrin is too big (280 kDa on 10% gel + a 30 kda..eek!). Discs large is also out. Suggustions please???
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4 comments:
I can't help you, but I just had to say - I actually know what a Western blot is!! Unlike the other lab science-y stuff that goes on in all these blogs I read :).
Ummm...not a fly person myself, but there are several master developmental genes that would be on in virtually all tissue...at least in imaginal discs and possibly adult tissue as well?
Dpp, hedgehog, Ubx? Maybe Ubx is best from that list? You should probaly check database for timepoints/expression patterns just to be sure.
I don't have any idea about relative abundance or decent antibodies...just pulling those guesses out of the cobwebs of my brain.
If you're really stuck try googling "laboratory protocols online"...I ususally get good suggestions there.
@PD - you go girl! I would not even pretend to know anything about coding or whatever it is that you do.
Another not as accurate, but fast thing you can do. Is stain your membrane with Fast green and see if a band is at the right molecular weight (if you have a lot of it, it'll be bigger).
Unfortunately, I know about as much as AA about flies. And AA took most of my suggestions. I'll think about it, though, and ask people in the lab when I get in.
PD - woohoo to knowing what a western is! that is impressive since I don't really understand anything other than unix linux and vista is crap, haha
AA - all excellent suggestions, I should've mentioned the other criteria is that it has to be a protein related to cell polarity as that would ensure we would have an antibody for the protein in our lab
amanda - i normally use ponceau red to stain the membrane, but I've read that the ponceau doesn't wash off cleanly so it can interfere with antibody binding. Since I want to confirm that my anti-vertrebrate protein is cross reacting with my invertrebrate protein, I want to minimize the possibility of it not working for any reason outside of not actually staining.
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