TCA Precipitation to remove albumin - (Aug/18/2012 )
I am doing a project with human serum, as i need to remove albumin., i tried TCA preciptation, according to a new protocol saying that TCA preciptation can reduce albumin content.. i used 4 volumes of TCA/acteone. after the entrie procedure i got a pellet. the problem is i tried to dissolve the pellet in 1M tris pH 6.8 but I cudnot dissiolve. After TCA perciptation i need to go for protein estimation by lowrys method then SDS-PAGE then 2D gel then mass spectrometry.. I have read tht we can dissolve pellet in 2X SDS-Loading buffer... But as i need to go for protein estimation by lowry's i don't think so i can go for SDS loading buffer... Pls do just me a method were i can continue through my entire project....
I would dissolve in RIPA buffer. It never affected my Bradford assay reading, even after addition of protease inhibitor.
It it were me, I would not use TCA to remove albumin, because it non-selectively precipitates all proteins. There are kits available for albumin depletion, which are used prior to 2D electrophoresis and I have heard they're quite OK.
As for dissolution of your pellet, I would go with SDS buffer and BCA or Bradford for quantification.
thanks for the suggestion but i have read an article that said it would reduce the content of the albumin present in the sample please do help me in this d suggesting a way out... like an affordable albumin removal kit.
There are a number of products on the market. Why don't you just do a Google search for a kit and try to find something that's within your budget. I know of kits from Qiagen and GE, but I don't know if they are within your price range.
I know about those Kits.... And most of them is beyond my budget.. That is when I found about this TCA precipitation...
dissolve in urea lysis buffer and bca assay it