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HELP_Bradford and protein problems - Different protein concentrations (Nov/10/2009 )

Purified proteins using CHAPS/Urea method for running 2D-DIGE (compatible buffer).
Need to quantitate using Bradford so final concentration of sample is 50ug/10ul.

HOWEVER, when I diluted my stock sample in water (as per the 2D-DIGE experts advice - they like to quantitate in water) to do the Bradford assay, it didn't appear to solublize well (got a clearish pellet in the bottom of the tube) and when I did the Bradford assay I got a protein concentration reading of say 10 ug/ul.

SO, I tried diluting my stock sample in PBS, and it appeared to dissolve much better, there was no precipitation or pellet in the tube, but I only got 1/2 the concentration (say 5ug/ul) of the EXACT same amount of sample.

I must have a final concentration of 50ug/10ul for the DIGE reaction to work properly. If I base my dilution on the readout from dissolving in the protein in water does this result in a 2-fold OVERestimate? Or if I base my dilution on the readout from dissolving the protien PBS does this result in a 2-fold UNDERestimate?

Do I go with the water or the PBS reading?
The PBS versus water does not affect the standard curve nor the Bradford dye.
It's just what stays soluble in my sample when i try to dilute it for the Bradford assay.

What would you do?
Has anyone encountered this?
Please advise!!

Oh - and if there is TOO MUCH protein in the final sample for DIGE, it will preferentially label the OVERabunant protiens and I won't get good results. If there is TOO LITTLE protein in the final sample for DIGE, the labelling reaction will not proceed well. Therefore, I really need the right range of protein!


Well the half raedings can be cuase u dint dilute the standards in PBS too!!! ideally the diluent for the standards and the sample should be the same to get a cmparble result. And if the pellet was not soluble completely i would not rely on that result anyways. Try solubilising it in GuHCl. Bradford gives no interference with GuHCl (unless it is in too much quantity). or try the micro bradford so that u dilute your protein more, thus solubilising more!!
Best luck!!

-Pradeep Iyer-