PARP antibody working condition - (Jul/04/2006 )
Does anyone have experience using PARP antibody to see the cleavage when apoptosis occurs. my question is: is this antibody need specific lysis buffer containg DTT before lysed the cells.
It depends on the Ab. You can find this info on the datasheet usually. Some people say you should lyse the cells in Urea to detach the PARP from the DNA but one of the cleaved fragments you want to see (the one around 80-90) is the one that doesn't bind the DNA, therefore I don't think one needs to use Urea. Moreover I've used a classical RIPA buffer to lyse my cells and detected PARP without problems, even the full size PARP
thank you. I try use normail lysis buffer but i did not obtain the pattern as published in other paper. I did see two band about 115 and 86. therefore i need to improve.
So you are doing a Western Blot??? Usually the datasheet should tell you if there are special things you need to consider. On what kind of cells are you using ab??? And what is your basic protocol??? It's easier to give advice or hints.