I need to extract total protein from duckweed plant for Western blot.The composition of the extraction buffer which I am going to use consists 50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 2 mM EDTA, and 2% PVPP. As I know PVPP is hard to dissolve and I was wondering if anyone has any idea as to how the solution is made? Can I use extraction buffer with insoluble PVPP to extract protein for immuneblot?
Any help would be greatly appreciated.
Thank you
Dear Prajani,
PVP is typically added to bind to polyphenols and thereby counter any negative effects in downstream applications. Many protocols for making plant extracts use PVPP for the same purpose. As PVPP is insoluble it will be in the pellet after spinning the extract and there should be no interference in immunoblot. It is good practise to pre-swell the PVPP before use. Also, you may want to make a quick experiment to compare if you need PVPP at all. If it doesnt give you any advantage (band resolution, sensitivity) you may simplify your buffer.
Cheers Markus
Prajani, I used PVPP this week. 2,5 % (w/v) of PVPP for the experiment. I found that PVPP make debris tighter, transfer of lysate easier.
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