[Arabidopsis] protein extraction from cambium

Hilary S. Callahan via arab-gen%40net.bio.net (by hcallahan from barnard.edu)
Thu Jun 25 09:12:09 EST 2009


Hello,

Here is a question from one of my students. It's not about Arabidopsis, 
but I would imagine that some readers of thi list have ideas about 
extracting from very low protein concentration?


QUOTE:

I am in Finland right now working as a trainee for 4 months (well 2 
months left to go). My project is looking at telomerase activity in 
different aged trees and also in different tissues of the same trees. 
I've been trying to iron out the protocol for the past few weeks...

We are taking protein extracts from needles, buds and cambium tissue 
types for this experiment by grinding tissues in liquid nitrogen and 
then resuspending in lysis buffer. The problem is, trying to collect 
protein from cambium yields extremely low quantities and we think this 
is because there is so much water content in the cambium compared to 
other tissue types. I have tried increasing the homogenized 
tissue-to-buffer ratio as much as possible and still barely manage 0.1 
µg/µl (target is 1.0 µg/µl) which means I need to either attempt to 
concentrate the protein or use a large quantity of this protein extract 
in my TRAP/PCR. I've tried both ways - concentrating the protein causes 
the lysis buffer also to become concentrated, which then causes issues 
with protein concentration assay as well as PCR, because lysis buffer 
has all these different noxious components in it... and with the second 
option, having large quantity of lysis buffer in the PCR also may have 
interfered with the results because when I ran the PCR there were no 
bands in the cambium lanes even though theoretically the amount of 
protein in the cambium lanes was approximate to the amount of needle and 
bud protein in adjacent lanes.

So now I am sort of stuck. My mentor and I both think that the best path 
forward is to look for ways to extract a higher initial concentration of 
cambium protein so that we don't have to deal with lysis buffer 
concentration issues in later steps, but I am not sure how to do this. 
So I am asking everyone scientific-minded that I know for suggestions! 
Do you know of any protocols that allow extraction of very low protein 
concentrations?



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