I am doing the click reaction on the HEK293T cell lysate (200ug proteins) for 1h at R.T. After the click I perform the MeOH-CHCl3 precipitation, then I try to re-solubalize the pellet in 4% SDS but it is not completely re-silubalized. Can help is appreciated regarding to do I efficiently re-solubalize the pellet after MeOH-CHCl3 precipitation!!.
I have also performed MeOH-CHCl3 precipitation. But then I used all of it for SDS-PAGE sample preparation (precipitate + 1xsample buffer then 95degC 10min). For you I would thing about some solution for solubilization of inclusion bodies. For example: First suspending the pellet in 4M urea, then mixing with equal volume of 4% SDS. But that depends on the constraints of you following work. Urea can cause modification of lysines and arginines. But there are ways how to suppress it (low pH + NH4 cations in solution).
Thanks @Peter. In my case I can't directly add SDS sample buffer to the pellet and boil at 95°C, because I need to pulldown biotinylated proteins by Neutravidin beads in the following step!!. (As I do the click with Biotin azide). I think 4M Urea should not hamper the biotin pulldown in the following step??.
I would try resuspending and dissolving the pellet in 6M urea and then diluting the solution to 2M UREA. If the proteins stay in the solution I would try the binding to neutravidin.
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