Rubsico Precipitation during extraction

ITQB jpmaroco at itqb.unl.pt
Wed May 9 08:58:39 EST 2001


Hi,
I am trying to extract Rubisco from grapvine leaves, and I generally end up with none. I use standard extraction buffers (200 mM Tris.Cl pH8.0, MgCl2, EDTA, etc.) and I have to use up to 10% insoluble PVPP and up to 5% Triton X100 to actually see some rubisco in SDS-PAGE gels.  I believe that the problem is that Rubisco is binding to phenols and its is being pulled down during the centrifugation step. Triton does help to solubilize some of this aggregates, however I do not get consistent results (sometimes works but most of the time it does not...). I believe it must have to do with plant's growing conditions (some leaves are more phenols than others). As anybody run into a similar problem? Any sugestions on how to solubilize Rubisco (in active form...) from whatever agregates its doing? Thanks a lot.
Joao 
**********************************************
João P. Maroco
Lab. Ecofisiologia Molecular - ITQB
Av.Republica. EAN
2780 Oeiras
Portugal
Phone: 214469641
Mobile:965447096
Fax: 214433644
e-mail: jpmaroco at itqb.unl.pt
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