hi all,
i am trying to concentrate some mammary proteins isolated using Trireagent for Western analysis. i have been using 3kDa Centricons from Millipore, which would normally work fine except for one hinderance - my concentration of SDS. My isolated samples are in 0.1M Tris-1%SDS, which (if my calculations are correct) is equivalent to 34.7mM SDS. The CMC of SDS is 8.3mM and the MW of an SDS micelle is 18kDa. Therefore not only would I be concentrating my protein, but also the SDS. To remedy this (so I thought), I diluted my protein samples 1:10 so that the SDS concentration would be less than the CMC. Long story short, I started with 1mL of diluted protein sample in the Centricon and I am now down to ~35uL, but the concentration is only just above my original (pre-dilution of 1:10) concentration, and still not enough to run a reasonable volume on a SDS-PAGE gel!

Is it feasible to just concentrate my undiluted protein isolates and not worry about my final concentration of SDS for WB?

Any suggestions would be greatly appreciated

I use 25% PEG8000 to concentrate my protein. briefly, add 1:1 Vol of 25% PEG8000 to the protein extract, RT 30min, 18000g or higher spin down.
I use this to concentrate my coprecipatated proteins, hope it will work for you.

I have ever used acetone 1:4 to precipatation the protein for western blotting. But I found after acetone precipatation, the non-specific can not be blocked even by 30 % skimed milk or 10 % goat serum in primary antibody incubation.

I am trying to find some reagent which can be use. Is PEG 8000 working? I dont know, hope it wont affect western bloting.

PEG 8000 (or higher) definitely works for concentration. The thing you have to look out for is that it's often contaminated with lots of other small molecules that leach into your sample.

Spin concentrators can also work well for some proteins, but other proteins aggregate and precipitate at the membrane.

I often go with the simple solution of just putting my protein in a dialysis cassette and letting the water slowly evaporate through the membrane while it sits in the refrigerator.

Where does these small moleculors come from? From PEG800 itself.

Can you give me the protocol PEG800 concentrating?

I cant use dialysis because the total amount of my protein sample is only 120 microliter.

Yes, PEG is often contaminated with lots of other things, I think it's because for many of its uses people aren't really concerned about purity.

The method I would use is to put your sample in one of these small volume dialysis tubes:

http://www.piercenet.com/products/browse.cfm?fldID=04010165&WT.mc_id=forum

Put PEG in a microcentrifuge tube and add just a little water to make it gooey. Then place the dialysis tube into the microcentrifuge tube so the membrane contacts the PEG. The PEG will draw water through the membrane, leaving your concentrated sample behind.

You could also use the evaporation method I mentioned using these same small dialysis tubes.