Protein precipitation with PEG 4000 - (Jul/16/2007 )
I'm trying to precipitate a 600kd protein using 16% PEG 4000. Usually it works great (about 95% protein precipitates) but sometimes, only 10% precipitates! I can tell immediately if it's going to work (the solution will be cloudy upon addition of the 50% PEG solution). I can't figure out what the variable is: the temperature of the sample (room temp or straight out of the fridge)? The way I add the PEG solution? Would these things make a difference? I've tried to be consistent with the starting material, I've tested old solutions of PEG vs freshly made solutions, buffer pH, etc. None of these things seem to be the culprit. Any ideas?
Protein concentration is a major factor. Ionic stregth, pH and temp are all important variables. Have you tried PEG8,000?
When I do the PEG precipitation, the starting material, in terms of buffer and protein concentration is generally the same. My starting material is clarified plant extract. I extract it the same way, in the same buffer, each time. Though I did do an old buffer vs new buffer test (didn't make a difference). Also, I've passed the clarified extract over tangential flow filtration to concentrate it 10-fold before precipitating with PEG. Again, sometimes 100% precipitates and sometimes 10%... This is could be a duh! moment: sometimes the air conditioning goes out in the lab and it could be 90degrees in there. So room temp is not necessarily the same each time. Even though the precipitation takes place on ice, do you think the temperature of the PEG solution itself plays a part in this?
Also, aside from this project, I've never used PEG before. I just found a paper where they used PEG 4000. When it works, it works beautifully. But, I should try PEG 8000 you think?
The question would then be 'How long do you keep the solution on ice?' Was enough time given for the mix to equilabrate temperature wise with the ice? And was the mixture shaken while it was ice?
Well, that's how this whole confusion started: I was getting 100% precipitation then one day I got about 85%. I figured it was because I had moved to a larger scale (in a 50 ml conical tube rather than a microfuge tube). I assumed incomplete mixing, surface area against the ice, etc. (at least 30 minute incubation by the way). So my next experiment was a small scale test (1ml in microfuge tubes) in ice, in ice + water, in ice + salt (lower temp) and ice but with a vortex every 5 minutes or so. The results from that was all only gave me 10% precipitation! (But that was the day that the a/c was out and it was 30C in the lab). Shrug.