Adventures in protein land

I’m not a protein biologist.

I wouldn’t usually describe myself as a geneticist either, unless trying to clarify what it is that I spend my time doing, in the same way that for years I would describe myself as playing the violin rather than as a violinist, because that seemed to indicate some level of skill that I didn’t feel I yet had.

But I’m definitely not a protein biologist, because I did no protein work as an undergrad, and I didn’t sign up to do a protein based PhD, and there’s only one post doc in the lab who has the first clue about proteins and – while she is possibly the most helpful post doc ever to cross my academic path – there’s only so much help someone can give you when you don’t know what questions to ask.

Yet in spite of this, I appear to be in a situation where doing protein work is inevitable. A few months ago I learned about PAGE gels and Coomassie blue staining and Western blots. Now, I must actually start extracting protein from real live plants, as opposed to my nice-n-easy-burst-like-a-bubble-E. coli. It turns out that this is rather more complicated than I had hoped. 

For starters, the shopping list is insane. I have had to buy:
– Glycerol (yes, I know the lab should have it. We don’t. I have no idea why)
– A protease inhibitor cocktail
– PMSF (which is mental-toxic)
– CHAPS (some crazy ass detergenet)
– Urea
– Thiourea
– Benzonase (still not really sure what this is)
– ASB-14 (likewise)
– Trichloroacetic acid

Plus a bunch of stuff we already had in the lab (KCl, Tris, the makings of phosphate buffer…)

That’s without me buying pharmalyte, which is crazy expensive, and which you need to run 2D gels. Except I’m not allowed to run my own, and the proteomics facility say they don’t do them any more, but if they did then they’d buy pharmalyte for me and … blah.

Basically: Dear diary, I had to spend a lot of money on stuff I might only use once. And then wait for it forever. And then wait longer because I forgot about the TCA. 

Now I have all the stuff, and I’ve started trying. It turns out that – counter-intuitively – there’s more that goes wrong with protein extraction than RNA or DNA. Or maybe I’m just not used to it. Soil for instance. In an RNA extraction you mix up your roots with Trizol, and then spin the hell out of it, and the RNA comes out on top. Simples. But in protein extraction there’s hardly any cytosolic fluid, and your membrane proteins go to the bottom… with the soil. Have you ever tried to get every last scrap of soil off a bunch of roots? Trust me, fun it ain’t.

Anyway, on that note, I should probably shuffle back into the lab and see what I can do with the last hour or so of the day!

3 responses to “Adventures in protein land

  1. Don’t worry to much about the soil. After homogenizing your tissue in the buffer and before spinning down filter your sample through a mesh filter, this will remove most of the debrish. Also when you extract your protein out of the pellet, if there is soil in this pellet it will stay in the pellet.

  2. Hmm, I’m sure it wasn’t this complicated last time I ran a gel, but I suspect my lab use a lot of expensive kits 😮

  3. Pingback: Adventures in protein land part 2: Cytosolic protein extraction | bakingbiologist

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