Protein TCA Precipitation Resolubilization Question - (Aug/24/2006 )
I am having trouble getting my TCA precipitaion pellets to disolve in SDS-PAGE sample buffer (2x or 4X + ME).
As a result I do not have enough soluble protein for detection with rather insensitive antibody.
Does anyone have any advice on how to increase solubilization of the pellet after TCA precipition of a cell lysate?
your harsh pH may be a problem if it is not compensated by your sample buffer (remains it yellow if using BPB?); some proteins such as some large cytoskeleton or myofibrillar proteins remain difficult to resolve; do not heat your samples in sample buffer, try to neutralize your probes, sometimes low amounts of DMSO increases solubility, vortex well
I have the same solubility problem. I votex 1 h, but the pellets are still there. I'm about to leave in +4 ovn. I wonder if it because I dry the protein too much under N2?
in my experience I do not let that the pellet dry to much, usually after the acetone washing I wait 15 min at RT then I add 2X leamly.