Trials and Tribulations
Don’t you just hate it when 3 days worth of work goes down the tube? Better yet, don’t you hate it when you follow a protocol (which you have successfully done twice before) to the letter, and you get still get nothing?
That’s basically what I endured yesterday. This Tuesday, I was running smoothly through my protocol for nuclear translocation assays (again testing for the presence of “Protein X”). I did everything that I had done before with this assay…starving the endothelial cells, restimulating them with a particular ligand, permeablizing their membranes so that antibodies can enter, stimulating them with the specific antibody for “Protein X”, and finally adding a secondary antibody with a fluorescent green tag. All stuff I’d done before.
Except THIS time, when I looked under the microscope…nothing. Absolutely nothing. No green glow from the fluorescent tag. Same problem for all 12 slides I look at. I won’t bother to put up a picture, because that’s what you would see…nothing. Blackness.
So yeah, it was a pretty frustrating experience, but that’s the best way to learn. After some troubleshooting, me and Nam concluded that the secondary antibody (the one with the fluorescent tag) was the problem. Or so we think. Tomorrow (Friday) after letting my cells grow, I’ll stick a different antibody on “protein X”, this one with the old red tag that I had used earlier.
It could have been the antibody. It could have been contamination. Or, it could have been me being careless. But regardless, like I said, messing up is definitely the best way to learn, even though it is pretty frustrating. The best way to get better at something is to practice, and this is no exception.
Til next time.
“The greatest mistake you can make in life is to be continually fearing you will make one.”
-Elbert Hubbard
