Thursday, April 5, 2018
Boring stuff Thursday #1 -- Not all Glu-C enzymes are the same.
SO...Here I was with evidence it was finally here. I'm insane. For real. Not "Oh, look at the old guy climbing a tree outside the bar, I bet his kids are embarrassed" crazy. The real one. Where you concentrated really hard and on the 5th time you still FAILED TO DO AN IN-GEL DIGESTION. You QC'ed the instrument again. And still nothing. Air bubbles in all the vials? You've checked everything and that help wanted sign you saw on the door at the gas station this morning strangely pops into your mind. What is that doing there? I'm trying to troubleshoot something. No random flashbacks!! Wait. Did I change a nanoLC column at 9pm last night? Why would that even make sense?!?
Wait. What's this? The Biopharma Finder runs you queued up last night finally finished (my gosh -- great software -- but we need to see if we can install it on a cluster or something -- slooooow....) says this other protein digested great with trypsin AND LysC, but no Glu-C peptides at all? It's not me (exclusively) me!?!? It might (also be) the enzyme!?!?
Okay -- so -- I'd just assumed everybody had the exact same enzyme source and they probably just change the label on the vial. Apparently not true.
Promega (don't sue me!) clearly states -- suitable for in-solution digestion. Doesn't say in-gel. Ask my friend who didn't take 5 years off from science. Of course she already knew this.
Okay -- crisis averted -- I guess. Especially since a friend can loan me one that is compatible tonight!
Pierce's Glu-C specifically states it is in-gel compatible.
NEB says theirs is and recommends a specific buffer they provide and protocol.
Roche Glu-C is in-gel compatible.
Probably others. And maybe Promega's is too, but it doesn't work in my hands. It worked great for an in-solution digestion, though.