Pitfalls of Protein Extraction by RIPA buffer
Protein extraction using RIPA buffer is a common practice. However due to the lack of comparable method, there is no direct comparison of protein profiles extracted by RIPA buffer and other methods. Here we report a side by side comparison between a rapid (1- 3 min) novel protein extraction kit and RIPA buffer.
Materials and Methods
Proteins were extracted from mouse liver and splenocytes by MinuteTM total protein extraction kit for animal cultured cells/tissues (referred to as Minute kit below) and RIPA buffer (R0278, Sigma). Briefly, samples using Minute kit were prepared according to manufacturer’s instruction.
There is no insoluble fraction with Minute kit. For RIPA buffer, tissues were homogenized and extracted for 30 min with shaking. The lysates were centrifuged at 12,000xg for 10 min. The residual insoluble fraction of RIPA extraction was further extracted using the Minute kit and analyzed by 12% SDS-PAGE.
