Responses to PVP/PVPP question.
hroychow at NMSU.EDU
Thu Mar 6 18:09:50 EST 1997
Thank you for all the responses to my query. All were helpful and generally pointed to the same direction some of us had originally surmised. I have not had the time to check out the references yet. In general, the idea seems to be the usefulness of PVPP in eliminating majority of the phenolics and polysaccharides etc. during the first centrifugation step. The PVP, on the other hand, acts through the successive steps continually (methinks). In my hands, both the processes seem to work well, but PVP-containing CTAB yields a slightly cleaner prep in the end.
Following are the responses I received today:
I do not mean to be a smart _______, but how about doing a small
experiment and determine which works best in your hands? Compare
recoveries from multiple samples using the two reagents? Look at
purity via gels, and A260/280 and even if it will PCR?
just some thoughts,
I have seen your posting in the microbio newsgroup. I do not think you
will find a ref for the comparison PVP / PVPP in the CTAB buffer. Use
either of these, knowing that PVP is soluble but precipitates with TCA
(problem when precipitating proteins) and that PVPP is insoluble and is
removed at the first centrifugation step. I hope this helps.
If you obtain some different answers, please communicate.
All the best
we use "insoluble" PVPP during the initial grinding of plant tissues (e.g., in
liquid N2, or with dry-ice in a coffee grinder). Supposedly it complexes with
phenolics that are released and oxidize during cell rupture. We use either a
Dellaporta style extraction buffer or a similar one with 8 M urea followed by
phenol extraction. In either case, the added PVPP is spun out during the
first centrifugation step. Sorry, but I'm not aware of an official reference
for the use of PVPP (or PVP) in NA extractions.
I don't have the original method, but I do know something about PVP and
PPVP. They differ in that the first is soluble in water while the second is
insoluble. Therefore PPVP is much easier to subsequently remove from the
extraction procedure. I have used PPVP extensively as an initial clean-up
step for the removal of phenolics, although not in a NA extraction. I
believe PPVP is used resonably frequently when purifying proteins from plants.
The question is one solubility. You choose the compound that won't get in
the way of what you are isolating. There must be a more recent ref, but
here is one from my thesis.
Loomis, WD (1974) Overcoming problems of phenolics and quinones in the
isolation of plant enzymes and organelles. Methods Enzymol. 31, 528-44
A lab manual prepared by Scott Rogers says that PVP (1%) is used in prep
of the CTAB buffer; this should be referenced in any of his chapters,
papers, I'm not sure which one eg Rogers and Bendich (1985) PMB 5: 69-76;
or Rogers and Bendich (1988) in Plant Molecular Biology Manual, Kluwer
Academic pp A6:1-10.
My recollection is that PVP is the soluble form is PVP
(Polyvinylpyrrolidone) and the insoluble form is PVPP
(PolyvinylPOLYpyrrolidone); both are used to "trap" interfering substances
(polyphenolics). The advantage of the PVP is that since its soluble you
have a known amount per volume of CTAB buffer (or any other for that
PVP may be considered an "optional" ingredient depending on the need for
it (presence or absence of problems caused by phenols)
See:Wang and Vodkin.1994.Plant Mol. Biol. Rep.12:132-145
Chang, Puryear, and Cairney.1993.Plant Mol. Biol. Rep. 11:113-116
Dr. Hiranya Sankar Roychowdhury
Plant Genetic Engineering Lab.
New Mexico State University
Las Cruces, NM 88003
Ph. (505) 646-5785
hroychow at nmsu.edu
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