So this is going to be more like a lab book entry than anything else, but I’m finally starting to get my head around some of this protein malarkey and figured I should write it down in multiple places!
My protein extraction protocol is more or less based upon Giavalisco et al (2003). I already gave you a shopping list for most of the ingredients.
Inhibitor buffer 1
0.0592g potassium chloride
1.6 mL glycerol
Plus one tablet of cOmplete ULTRA (EDTA-free)
This makes way more than you need, but goes off pretty quickly. I’ve been freezing aliquots and it doesn’t seem to have made any difference, though if you’ve got the money to do it fresh every time then I would.
My protocol helpfully calls for 8.75 uL of Phenylmethanesulfonyl fluoride in 12.5mL of 100% ethanol, which is pretty annoying given PMSF comes as a powder. A lot of internet searching assures me that a final concentration of PMSF of 1mM is about the right ball park, so I ignored the specific volume instructions in the protocol, and just made up 4mM in ethanol and used about 0.25 v/v. Again, seems to work so far
0.8g CHAPS (NB this stuff is fairly expensive, and sold by the gram)
[Pharmalyte if you're doing 2D Westerns, but I'm not and it costs stupid amounts so I don't add it]
Water to 20mL
Flash freeze plant tissue in liquid nitrogen and grind to a powder. While still powdery at 0.125 parts of inhibitor solution. (Realistically, I have added an excess every single time, because that is so little and there is so much tissue. I’m probably being a bit wasteful here, but just FYI). Then add
0.05 parts PMSF to a final concentration of 1mM.
Centrifuge the goo at 4C for 1 hour. After this the goo will separate into a supernatant containing cytosolic proteins, and a pellet containing membrane proteins. And soil. Even after you’ve washed the roots for an hour. Sigh.
Remove the supernatant and add 0.5 v/v of the lysis buffer. Freeze at -80C until you’re ready to clean it up. (With roots I get about half as much liquid out as I put in, but that’s partly because the roots aren’t quite ground to powder, so they retain some liquid as they don’t form a pellet properly).
When I write it down it looks very simple :-/