I have used the methanol/chloroform purification method to purify proteins many times with good results. Last week, however, this method purified proteins pellets (from total protein isolated from a mouse cell line) that I have not been able to dissolve it in anything, including 8M urea, 2% SDS and other similarly harsh conditions. Do you have advise how to dissolve them? What factors might contribute to the formation of such insoluble protein pellets?
Hey Nikolai, just for my own education first, the methanol/chloroform is to get the protein for what? do they generally remain folded/functional under the conditions or concentrations you use?
How about DMSO, acetonitrile, etc to dissolve? Or formic acid
I was using methanol/chloroform method few times. You should watch not to overdry the pellet.
But the most effective way of dissolving the pellet was sonication. Try it, I am quite sure it will help. I was dissolving the pellet in 1X sample buffer for SDS-PAGE.
@Toufic el arnaout: proteins do not remain functional, they are denaturated.
Hi Dawid, thanks. Yeah I know but was addressing the fast that he called the other conditions "harsh". I mean proteins are already denatured and went through strong organic solvents, why to worry about if they are "harsh"..
Sonication should work. Otherwise you could also try adding a little Triton X-100 or Tween.
Thank you Toufic and Dawid,
The protein prep is for mass-spec and the proteins are likely denatured during the extraction procedure which involves both urea and SDS. I did try DMSO, Triton X-100 and continuous sonication at 35 C. I also tried dissolving the pellet before I have dried completely the methanol. All these helped a bit but they did not dissolved the pellets sufficiently to be processed further for trypsin digestion.
Hi Nikolai,
I am in the same situation of having an highly insoluble pellet after chloroform methanol extraction. The extraction buffer I used had 8M Gn-HCL in it and samples are prepared for mass spec. Did you have any luck in finding a solution to this problem?
Thanks
I followed the same protocol for protein precipitation by Weesel & Fluegge
And finally dissolved the pellet in 6M Urea made in Tris buffer, pH 7.8
And gently tapped the tube and kept it for 5min in 37*C
Although it worked for me in case of pellet solubilization but I'm not sure about the protein loss.
Will determine the concentration by tomorrow.
Hi, Anjali Patni ... Can you tell me about the protocol you used for dissolve proteins? Is it works? what about the proteins loss?
Thank you!
Usually 1M NaOH works well for solubilizing methanol-chloroform precipitated pellet. Once the pellet is dissolved you can gradually adjust the required pH.
I have resuspended my pellet in 6M GnHCl followed by 60oC incubation (10-15 mins). Then diluted the Gn to
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