[RASMB] Re: peculiar leakage problem!

[John Philo]

Sumit, one thing you haven't clearly told us is whether the leaks are
occurring via the fill holes or between the centerpiece and the windows. If
you are not consistently getting proper sealing at the fill holes then a
cell that didn't leak when filled with water might leak after re-filling
with a protein sample. 
 
Also remember that one way a protein sample is not equivalent to water is
that the protein itself can substantially reduce the surface tension and
allow wetting and leakage through centerpiece defects that would otherwise
not occur. I find it is much harder to seal samples containing detergents or
those at very high protein concentrations because the surface tension is
low.
 
John

  _____  

From: rasmb-bounces at rasmb.bbri.org [mailto:rasmb-bounces at rasmb.bbri.org] On
Behalf Of sumit goswami
Sent: Friday, June 12, 2009 3:47 PM
To: rasmb at rasmb.bbri.org
Subject: [RASMB] Re: peculiar leakage problem!


If I allign the cell properly wouldn't I expect the screw hole to orient
towards the center properly?
I indeed didn't mention what material I used. I used charcoal filled epon
centerpiece. Recommeneded speed 42000rpm but we routinely use it at 50000rpm
like many other people. But the speed doesn't solve it as the same cell
assmebly that didn't leak with water leaked with the protein sample the very
next time.
 
Sumit


 
On Fri, Jun 12, 2009 at 4:51 PM, sumit goswami <ikant11 at gmail.com> wrote:


Dear all
            we have been trying do some sed-velocity experiments for last
couple of weeks but every time we tried most of the cell assemblies leaked!!
Apparently it seemed like a simple problem of aging of center pieces but
then very recently I ran all the seven cell assemblies overnight at 50000rpm
with water in them and none leaked! After that I used the same set of
assembly for running my sample by sucking off the water from them  and 4 out
of 5 leaked even at 4000rpm. We have used different kinds of buffer so
far... PE (5mM Na-phosphate, 1mM EDTA), TE (10mM Tris, 1mM EDTA), Poly
buffer (MES,tris, acetate). All of our buffer had 0.01%PEG in them. We have
tried both PH 5 & 8.
   Can anyone suggest what's going on? We are ready to buy new centerpiece
but not sure if that's the problem. The result with water is confusing us!
 
Thank you very much!
 
Regards,
Sumit 


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