anthocyanins and protein isolation

Peter Gegenheimer peterg at rnaworld.bio.ukans.edu
Tue Aug 30 16:31:40 EST 1994


In <33tc37$kn9 at mserv1.dl.ac.uk>, "Nokwanda Makunga" <MAKUNGA at gate2.cc.unp.ac.za> writes:
>I am currently working on isolating total protein from 
>callus cultures of a anthocyanin pigmented line as well as a
>non-pigmented line from the same plant species. My extraction
>buffer has protease inhibitors (PMSF, EDTA) included in it and
>contains 3,5 microlitres per 10ml of mercaptoethanol . After
>extraction I perform a biorad assay to quantify protein isolated.
>I generally observe that yields are a lot less for the pigmented
>line. I then run 1D and 2D PAGE gels using apparently equal
>quantities of protein for both lines. Bands obtained for the 1d
>gel (red callus) are always fainter or a smear as compared to the
>white. For the 2d gels only 1 or 2 spots are obtained with the
>anthocyanin-rich callus meanwhile the white yields plenty of
>spots as expected. I tried to get rid of the pigment by running
>SEPHADEX G25 columns and I concetrate my proteins by dialysing
>against sucrose. I have also tried acetone precipitation  but I
>still obtain the same results.
>
>I would like to know  if anyone has had similar or the same
>problem and how did they overcome it.
>
>N.P. MAKUNGA
>DEPT .OF BOTANY
>UNIVERSITY OF NATAL
>PRIVATE BAG X 01
>3209
>
>E-MAIL: Makunga at AGRIC_PMBUNP.AC.ZA.

The first thing to try is to add either polyvinylpyrrolidone (PVP) or 
polyvinylpolypyrrolidone (PVPP). These will remove tannins but perhaps not
monoflavonoids like anthocyanidins. Gel filtration on Sephadex G25 or G50 should work
-- monitor protein and color separately. You may want - or need - to try a column 
with smaller pores, like BioGel P10 or P6-DG. Most of what I know was described in 
Meth. Enz. 182, 174-193 (1990) and references therein.

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