Sunday, November 1, 2009

Western Blot Woes (And Happy Endings!)

Not an EMT post to start, but at least this lab one has a happy ending!

To preface this post, one of the fellows in a neighboring lab told me this when I was trying to work this assay out months ago. “For a technique that is so commonly used in biology to prove ideas, it is by far the most fickle technique used.”

Day 1: Extract protein, quantify it, prep it, load it on a gel, get it to a membrane, incubate it with antibody to find protein of interest. A day of far too many samples with so little reward. I can’t even tell if I have screwed anything up yet!

Day 2: Finalize antibody stain to visualize protein with fun florescent molecules. After a bit of trial and error, produce a good looking film (yup, its just like x-ray film, but without the x-ray part. Photo sensitive instead).

But there is a problem. The antibody for my protein has previously, and consistently, been giving me only one band when I do this technique. Now I have two bands, at least about the same size (which is key), but two bands nonetheless. The difference between this run’s source of protein and the previous run’s source of protein: minimal.

I haven’t the foggiest why I now have two bands at the right size.

Day 3: Repeat staining process with a control protein antibody. Prep and visualize. Perfect film. Well, at least I know my technique isn’t off!

Day 4: Quantification. After imaging films, followed by far too much thought, and a fair amount of math, pretty graphs get to be formed! And on top of the fact that graphs in and of themselves are pretty, IT DID SOMETHING! My assay, done months ago with the cells saved in a -80°C freezer and a week spent working on the same Western Blot, did something!

I cannot begin to tell you how happy this makes me! Particularly since this means we may now have a mechanism for one of our models, and having a mechanism can be just the thing that is needed to get a paper published!

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