Resuspension of Triton-insoluble proteins for SDS-PAGE - (Jul/13/2002 )
I am isolating Triton-insoluble and soluble proteins from mammalian cells, yet I cannot get the insoluble protein pellet to re-dissolve in anything, like TNE, TNE with Triton or the SDS-PAGE loading dye/buffer. This is a nightmare for loading onto the gel. Anyone got any ideas?
Try SDS to 1% with increasing concentrations of DTT or some other reducing agent and heat to 70C. Are there many cys residues in your insolubles?
Try placing your samples in SDS loading buffer, then in a sonicating bath. It worked for a friend of mine.