Rehydrating/Resuspending Drosophila Proteins - (Oct/10/2011 )
I am using Biorad's ReadyPrep Sample Clean-up kit to cleanup my drosophila proteins before labeling them with ICPL technique. The protocol is for 2D sample clean-up but I am not doing 2DGE. I have to do SDS-PAGE after sample clean-up and isotope labeling.
My problem is that the protocol states we need to use the rehydration buffer from Biorad to resuspend the proteins and if I want to do SDS, I can resuspend in Lamelli Sample Buffer. However, I am doing SDS only after labeling my proteins with isotopes and if I use the Rehydration buffer from Biorad it might interfere with labeling since it contains Bromophenol blue designed for 2DGE. So, I thought of using 10 mM Tris.Hcl ph 8 as my resuspension buffer. I tried vortexing for long time and vigorous pipetting to break up the protein pellet but it is not dissolved/resuspended yet. Does anyone know how to dissolve the drosophila proteins after sample clean-up stage?
I would be really grateful for the answer.
Isn't the Biorad 2D rehydration buffer Urea plus detergent based? Once precipitated, hydrophobic proteins prepared by this sort of buffer would not be easily dissolved by Tris buffer.