Whimper

I spent two weeks on a complex lab procedure that was theoretically supposed to yield a visual representation of my success at isolating lactate dehydrogenase (LDH) from raw chicken breast muscle.  The process is a SDS-polyacrylamide gel electrophoresis (SDS-PAGE) procedure followed by Ponceau dying and then a Western Blot.

The idea is that the SDS-PAGE procedure separates the molecules you might have in your sample by size and, knowing the molecular weight of LDH, you can locate where it migrates to in the gel.  Then, you transfer the protein to a membrane and add the Ponceau dye which is supposed to allow you to see bands where various size molecules have migrated into the gel (which you have transferred to the membrane).  But then you remove the dye and add two antibodies.  One that binds specifically, and theoretically, only to your LDH, and the other binds to the first antibody and a chemiluminescent substrate.  The membrane can then be put in a special machine that can visualize the luminescent tag on the second antibody to prove the bands you see in your Ponceau dye are actually the LDH you hope to find.

Have I lost you yet?  Don’t worry.  I don’t understand it much either.

And this is proved by the fact that I just got my images back and have to write a report by noon tomorrow on this:

This is my Ponceau image of my membrane.  See all those bands?

Me neither.  There should at LEAST be one band in each row.  But as there’s no bands at all I can’t even tell where the rows are.

And this is my Western Blot:

Yeah.  So all those black smudges highlighted in purple?  Garbage.  There should be just one smudge in each of lanes two through seven.  So, maybe lane 7 has some LDH.  Or maybe not because obviously something is wrong as I have way more going on here than I should.

I just looooove when I put hours of work into something that didn’t work and I have no time to figure out why before I have to turn in a paper on it.

97 days of class until graduation.  97.