[ccp4bb] TCA or acetone precipitation of proteins
Hi Everyone, Sorry for the naive and non-CCP4 question. Is it possible to precipitate proteins (TCA, acetone) from a sample that has already been stored in protein loading dye? The protein is too dilute in my current sample and I basically want to load all of the sample (100uL) in a single well in the gel. Unfortunately, I already added protein dye with SDS and all. Cheers and thanks. Raji -- -- Raji Edayathumangalam Instructor in Neurology, Harvard Medical School Research Associate, Brigham and Women's Hospital Visiting Research Scholar, Brandeis University
Re: [ccp4bb] TCA or acetone precipitation of proteins
Are you sure that 100 uL won't fit in your loading well? Commercial 10x10 cm minigels have quite large loading wells. I think it's possible to squeeze as much as 50 uL into a standard 10-well, 1 mm gel. If you are using typical discontinuous SDS-PAGE with a stacking gel the sample will be concentrated to a tight band shortly after applying running current. While you may be able to precipitate your protein with TCA, incomplete precipitation and losses on resolubilization may not help you as much as you would like. Small centrifugal filters may get you down to 50 uL dead-stop volume, but that might be enough to load into a single well on a standard gel. A possible option is to add some dry BioGel with a low exclusion volume (e.g., P-6) but this will be tricky to get just right with such a small volume of sample. I've done this kind of volume reduction successfully with larger samples (1-2 mL), before there were centrifugal ultrafilters. Cheers, ___ Roger S. Rowlett Gordon Dorothy Kline Professor Department of Chemistry Colgate University 13 Oak Drive Hamilton, NY 13346 tel: (315)-228-7245 ofc: (315)-228-7395 fax: (315)-228-7935 email: rrowl...@colgate.edu On 9/9/2011 8:57 AM, Raji Edayathumangalam wrote: Hi Everyone, Sorry for the naive and non-CCP4 question. Is it possible to precipitate proteins (TCA, acetone) from a sample that has already been stored in protein loading dye? The protein is too dilute in my current sample and I basically want to load all of the sample (100uL) in a single well in the gel. Unfortunately, I already added protein dye with SDS and all. Cheers and thanks. Raji
Re: [ccp4bb] TCA or acetone precipitation of proteins
Is it possible to precipitate proteins (TCA, acetone) from a sample that has already been stored in protein loading dye? The protein is too dilute in my current sample and I basically want to load all of the sample (100uL) in a single well in the gel. Unfortunately, I already added protein dye with SDS and all. Methanol/chloroform precipitation is both more effiecient than TCA or acetone and it can do exactly what you want it to do. Wessel, D., and Fugge, U.I. A method for the quantitative recovery of protein in dilute solution in the presence of detergents and lipids. Analytical Biochemistry, 1984, 138:141-143 Or google it - there are many online protocols. - Dima
Re: [ccp4bb] TCA or acetone precipitation of proteins
Is it ok for you to heat your sample? If so, you could near-boil your sample in a heating block, say at 95 degrees C, in order to concentrate it. I've done this in regular 1.5 mL eppendorf tubes with either the cap open or where I've punctured the top of the cap with a narrow bore needle (in order to relieve vapor pressure from water evaporation). At 95C, reducing 100 uL down to ~20 uL probably will take 15-30 minutes. If you're working on a membrane protein, this is probably not a route you can take. Alternatively, maybe a speed vac will work? HTH- Brad On Fri, Sep 9, 2011 at 8:57 AM, Raji Edayathumangalam r...@brandeis.eduwrote: Hi Everyone, Sorry for the naive and non-CCP4 question. Is it possible to precipitate proteins (TCA, acetone) from a sample that has already been stored in protein loading dye? The protein is too dilute in my current sample and I basically want to load all of the sample (100uL) in a single well in the gel. Unfortunately, I already added protein dye with SDS and all. Cheers and thanks. Raji -- -- Raji Edayathumangalam Instructor in Neurology, Harvard Medical School Research Associate, Brigham and Women's Hospital Visiting Research Scholar, Brandeis University
