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.

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 

Hi Nikolai and Prathiba,

I also have a Problem with the insoluble Protein pellets like your now. I tried to dissolve it in SDS buffer, also DMSO. Both not working.

Do you have a solution now?

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!