Does anyone have any experience de-phosphorylating proteins in a cell lysate before SDS PAGE and western blotting?
I have a calf intestinal phosphatase, however I have heard that lamba phosphatase might be better. My main issue is that my cell lysates aren't massively concentrated, so if I dilute them in the buffer necessary for the enzyme, the proteins are then not concentrated enough to be used for a western blot?
Is there any way I can concentrate my cell lysate perhaps?
I would suggest treating with the phosphatase first, then concentrating the extract.
One way to concentrate it is precipitation with trichloroacetic acid (TCA) and centrifuge. The pellet can then be dissolved in SDS-PAGE sample buffer. This technique will precipitate the protein but leave most of the buffer and salts behind. Any residual TCA should be thoroughly removed, taking care not to lose any of the pellet. When you add the SDS-PAGE sample buffer with blue pH indicator dye, the dye should stay blue, not turn yellow.
Another way is to evaporate the sample using a freeze dryer or Rotovac. This concentrates everything in the sample that isn't volatile, including the buffer and salt, which can be troublesome for SDS-PAGE, so don't overdo it
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