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<DIV dir=ltr align=left><SPAN class=549472300-13062009><FONT color=#0000ff
size=2 face=Arial>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. </FONT></SPAN></DIV>
<DIV dir=ltr align=left><SPAN class=549472300-13062009><FONT color=#0000ff
size=2 face=Arial></FONT></SPAN><SPAN class=549472300-13062009><FONT
color=#0000ff size=2 face=Arial></FONT></SPAN> </DIV>
<DIV><SPAN class=549472300-13062009><FONT color=#0000ff size=2 face=Arial>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.</FONT></SPAN></DIV>
<DIV><SPAN class=549472300-13062009><FONT color=#0000ff size=2
face=Arial></FONT></SPAN> </DIV>
<DIV><SPAN class=549472300-13062009><FONT color=#0000ff size=2
face=Arial>John</FONT></SPAN></DIV>
<DIV dir=ltr align=left><BR></DIV>
<DIV dir=ltr lang=en-us class=OutlookMessageHeader align=left>
<HR tabIndex=-1>
<FONT size=2 face=Tahoma><B>From:</B> rasmb-bounces@rasmb.bbri.org
[mailto:rasmb-bounces@rasmb.bbri.org] <B>On Behalf Of </B>sumit
goswami<BR><B>Sent:</B> Friday, June 12, 2009 3:47 PM<BR><B>To:</B>
rasmb@rasmb.bbri.org<BR><B>Subject:</B> [RASMB] Re: peculiar leakage
problem!<BR></FONT><BR></DIV>
<DIV></DIV>
<DIV>If I allign the cell properly wouldn't I expect the screw hole to orient
towards the center properly?</DIV>
<DIV>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.</DIV>
<DIV> </DIV>
<DIV>Sumit</DIV>
<DIV><BR><BR> </DIV>
<DIV class=gmail_quote>On Fri, Jun 12, 2009 at 4:51 PM, sumit goswami <SPAN
dir=ltr><<A href="mailto:ikant11@gmail.com">ikant11@gmail.com</A>></SPAN>
wrote:<BR>
<BLOCKQUOTE
style="BORDER-LEFT: #ccc 1px solid; MARGIN: 0px 0px 0px 0.8ex; PADDING-LEFT: 1ex"
class=gmail_quote>
<DIV>Dear all</DIV>
<DIV> 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.</DIV>
<DIV> 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!</DIV>
<DIV> </DIV>
<DIV>Thank you very much!</DIV>
<DIV> </DIV>
<DIV>Regards,</DIV>
<DIV>Sumit </DIV></BLOCKQUOTE></DIV><BR></BODY></HTML>