I'm guessing you're looking for proteins in your sample ? If so I could recommend you solubilize in 8 or 6M Urea ( I use 6M Urea with 2M Thiourea) .

Well, just as it was mentioned by Tanveer, try to solubilize the protein pellet in urea-containing buffer. This will help You to get as much protein as possible. Sometimes it takes a while to dissolve the resulted "lump". Take your time.

Or try to use the DeStreak Rehydration Solution from GE. I was told that this reagent can halp you to get really sharp 2D gels. And remember to optimize protein concentration.

can u tell me your source of protein sample. As we devised single protocol for bacterial, yeast, tissue (mammalian), cell lines. So please let me know ur protein sample i will definitely solved problem

Dear Batth and Wieczorek,

I have already solubilizied the protein in rehydration buffer containing 8M urea before focusing.

Dear Kumar,

This was environmental sample (spring sediment). We could assume presence of proteins of both prok and euk origin. Basically this is a metaproteome analysis. But I guess there is all extracellular protein as I have not performed any treatment for the isolation of intracellular protein.

See if this paper can help you

Dear Sepehrimanesh and Kumar,

Thank you for your valuable suggestions. I will try all and let you inform the status.

With best regards,

Abhijit

Dear Abhijit

I have done protein isolation and purification by using denaturing condition.

So, whatever the sample, centrifuge it at speed sufficient enough to pellet organisms (say prokaryotes and/or eukaryotes); and then discard the supernatent and resuspend the pellet in SDS-PAGE sample buffer followed by sonication and heat denaturation. use this sample for SDS-PAGE. for purification purpose you can use denaturing solublizing buffer (50mM Tris, 300mM NaCl, 8M urea) as resuspension buffer followed by sonication to disrupt the cells and release the protein. centrifuge and process the supernatent for purification.

regards

Ramesh Kumar, IVRI bangaluru

Dear Abhijit:

For microorganisms sample preparation I use sonication or sample beads disruption, resuspending cells in a Tris HCl 20 mM pH8.8 buffer. After disruption, centrifugue samples and discard pellets. I always use the 2D Clean Up kit from Ge Healthcare to clean sample prior IEF. Its improve the quality of your 2D gel.

Regards

Laura

Hope you can see good seperation if you can follow the below steps.

1) Over night acetone precipitation in 4 degrees.

2) Dissove the pellet in re-hydration buffer.