Maybe you should try dialysis after electroelution to remove salts and SDS and then concentrate the sample.

 After the solubilisation you can try to run and small SDS-PAGE, could be that not all the pellet is protein. In the precipitation, do you see a in the interphase a white film?

Thanks Jaroslaw and Gabriela, I saw the thin interphase layer. As the salt is supposed to be dissolved in the top layer of methanol/ water, I believe the pellet is mainly built by the protein. I may try higher dose of SDS/DTT followed by dialysis. Hope that helps. 

Hi Shohreh, 

Just wondering if you found a solution to this problem. I am in the same situation of having an highly insoluble pellet which doesn't dissolve in any buffer, 50%TFE, 50mM DTT and  8M urea. Please let me know if you have an answer for that.

Thanks 

I use this method frequently. The only times i have ever had a completely insoluble pellet have been when i overdried.

When thats happened, i’ve had to resort to harsh temperatures, sonication, high detergent concentration. Even then, i lose a considerable amount.

I use this method frequently. The only times i have ever had a completely insoluble pellet have been when i overdried.

When thats happened, i’ve had to resort to harsh temperatures, sonication, high detergent concentration. Even then, i lose a considerable amount.