I'm using RIPA as a lysis buffer, but I have to concentrate my samples (primary HUVEC) due to low quantity of proteins. I concentrate them with filter centrifugations but this method also concentrates the detergents of the buffer. I'll use 1 mL of RIPA per sample and I need to concentrate them until 50 uL or so, so the detergents get REALLY concentrated. Is this a problem for SDS-Page runs for WB?
Why not try this:
Detach the cells first (e.g. using trypsin), spin the down, wash 2x with PBS, remove the supernatant, quick freeze the cell pellet, add the amount of lysis buffer you need, mix well, centrifuge cell debris at 16000xg and take the lysed protein supernatant for analysis.
It could be that 50 uL is not enough dependent on how many cells you have.
Another way to concentrate the protein in a dilute sample for SDS-PAGE is to precipitate it with trichloroacetic acid (TCA). Add 1/10 volume of 72% (w/v) TCA and 1/10 volume of 0.15% (w/v) sodium deoxycholate. Keep the sample on ice for 10 minutes. Centrifuge the sample at top speed in a microcentrifuge for 10 minutes. Remove the supernatant thoroughly* and dissolve the pellet in SDS-PAGE sample buffer.
* A method to thoroughly remove the supernatant without disturbing the pellet is to use gentle suction from a vacuum line through a pasteur pipet that has been drawn out in a flame into a fine point. A long, thin gel loading pipet tip can also be used.
One possibility is to make a more concentrated lysate by pelleting the cells and suspending them in a minimal amount of lysis buffer.
Alternatively, use chloroform/methanol to precipitate the protein (doi:10.1016/0003-2697(84)90782-6). The resulting protein film is relatively easy to bring back into solution, and there is nothing to interfere with PAGE.
It may not be a problem for PAGE and further WB if the RIPA is in use. If you have concerns and UF is the only choice for concentration, you may add urea-washing steps to the UF process (e.g. FASP) to sequentially removing of the detergent monomers. Breaking CMCs of the RIPA detergents and subsequent removal by serial washing may keep your protein enriched and also free from detergents. (BTW, always keep in mind the solubility issue for your target proteins both during and after the removal of the RIPA detergents)...