Hi all,
I'm going to use Pierce mitochondrial isolation kit (cat no: 89874) to separate cytosol protein and mitochondrial protein for WB.
1. For lysis of mitochondrial, instructions provided recommend to use 2% CHAPS/TBS, but could anybody knows if RIPA buffer is ok?
2. Does anybody know the supernatant containing cytosol fraction got from isolation process could be quantified directly with BCA assay and WB after SDS sample buffer? Does it need additional purification or lysis?
Thanks a lot!
I do not recommend to replace CHAPS/TBS with RIPA, as lysis/extraction conditions are different and therefore, it might affect yield/purity of corresponding fractions. Fractions can be normally analyzed with SDS buffer, but it depends on the characteristics of your protein(s). If you use Phosphatase inhibitors, the BCA method does not work. Try a 660 nm method from Thermo or another one. A table for compatible buffers/vompounds with the BCA method is attached. Check it as there is a dose effect of potential agents present in your buffers and cultured cells that could affect your experimental design.
Bon courage
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