1 reply to this topic

[chance3349]

Hi,

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.

[microdexter]

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.