how to prepare 200 mM HEPES/KOH pH 7.8 please help me - it is also containing 3 mM EDTANa2.2H2O, 3 mM magnesium acetate, 10 mM dithiothr (Apr/27/2006 )
PVPP is meant to be insoluble and once you've extracted the protein you get rid of it during centrifugation. Just make sure the PVPP is evenly suspended when you pipette it to the tube with your plant tissue (putting it on a stirrer works fine). It's also important to hydrate the PVPP for at least two hours (I usually leave it overnight so I can start straight away when I get to the lab in the morning).
From the Sigma site "PVPP complexes with phenolics and alkaloids for their removal from plant samples, thus preventing their modification of proteins and any interference they may cause in spectrophotometric determinations of protein content. This is also reported to improve stability of enzymes."
I notice you said 1% PVP (polyvinyl-pyrrolidone) before. PVPP (polyvinyl-polypyrrolidone) is different.
What plant tissue you work with will influence your extraction volume. I work with strawberry fruit and leaves. For fruit 2 volumes of buffer work well (i.e. 0.1 g tissue in 0.2 mL extraction buffer), but with strawberry leaves I use 10 volumes (0.1 g tissue in 1 mL) otherwise my extract is like a jelly. The best thing to do is to try using 2 volumes and if that doesn't give a "watery" extract, then try increasing to volume to 5 then 10.
What plant tissue are you working on? I can ask around if any of my lab mates have experience with it.
well i think it's surprising as PVP soluble at 2% in denhardt's buffer. I just put the powder and vortex till dissolution.
if you meant PVPP see previous post.