Removal of (pigments, lipids, DNA) waste

Savita Shah spshah at stanford.edu
Wed Nov 29 12:04:25 EST 2000


Hello :

I was working with insect digestive proteinases
and the problem was lots of poly phenols, pigments
and fat bodies in the extract which was
interfering with the proteinase assays.. I used
hexane to de-fat (and at the same time taking care
of the pigments).. Atleast in my case, there was
no loss in the proteinase activity.

I used following method:

Grind tissue in Liquid nitrogen..  lyophilize your
tissue and then pack in the syringe column (do not
forget to plug the syringe with glass wool or a
filter). Wash the column with several volumes of
hexane.. you will see the pigments and fats in
first few washings.. wash at least with 10 volumes
of hexane or until you do not see any pigments in
subsequent washings. Remove the material from
syringe in a plate and air dry.. You could then
proceed with regular extraction methods for
lyophilized samples. 

Extract the proteins in extraction buffer,
centrifuge and  collect the supernatant and treat
it with 0.1 to 0.05% Polyethleimine (spelling!)
..on ice for 30-45min. This will ppt DNA.
Centrifuge once more and continue with your
purification protocol. you see, you have taken
care of lipids, pigments and DNA!!!! 

The procedure seems to be time consuming
(especially the lyophilization), but I think it is
worth it. 

Hope this helps, Good Luck

Savita

Gaur rajneesh wrote:
> 
> Hi ,
> I am working on the purification of the scFv fragments of plant leaves
> origin.Does anybody suggest how to get rid of wastes such as pigments, DNA,
> lipids,preferably in one go without affecting the property of the protein
> solution.
> 
> I would appriciate the help.
> 
> Rajneesh,
> Ph.D. student
> Institute of Biology 1,
> RWTH aachen,
> Worringerweg1, D-52074,
> aachen, Germany
> 
> raj11 at bio1.rwth-aachen.de
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