[RASMB] Re: peculiar leakage problem!

[Leech, AP]

Hi Sumit,

If I am worried about the surface of the centrepiece, I put a window
on top of it (not assembled in the cell), and I can see the fringe
patterns, which should be even and not distorted. This is effectively
checking the flatness of the centrepiece surface with the window as
a standard. We have some old centrepieces where you can't see a fringe
pattern at all, and these are really prone to leaking.

I assemble the cells and tighten by hand initially, and if I can't see
a fringe pattern under the window I disassemble and check for dust and
fibres. Then I tighten up with the torque wrench and the window to
centrepiece contact area should go black - then check for fibres and
dust again which sometimes show up at this point.

If your filling hole gaskets and screws are not good and you put the
rotor under vacuum without spinning, liquid will come out of the cells.
I know there are reasons not to, but I prefer to spin the rotor at low
rpm at least until I'm happy the cells aren't leaking - the spin keeps
the liquid away from the fill holes and it is easy to see the meniscus
moving if it is a bad leak. The live display on the interference optics
is very useful for checking this, but single scans in absorbance
(collect in intensity mode) will do.

Check your cells carefully when you disassemble them and clean off any
salt etc as it is rather corrosive to the aluminium and may make it
difficult to get centrepieces or filling hole screws in place, which
will not help with getting a good seal.

Hope this is useful,

Andrew

sumit goswami wrote:
> For sure I put the cell inside the rotor with the holes facing towards 
> the axis of rotation. But I am not exactly sure if the leakage occured 
> through the feeling hole or between the centerpiece and windows. I have 
> seen spots of dried sample on the ouside surface of windows but I don't 
> remember seeing any circular line at the wall of the centrifuge chamber. 
> I have seen marks of leaked sample at the bottom of the rotor where it 
> supports the cell. So in my understanding it is very likely that the 
> leakage has occured between the windows and the centerprice. One thing I 
> must have to say is that our centerpices are old and they might have 
> some scratches or damages that are not clearly visible through naked 
> eyes. As Dr. Philo mentioned it might be that in presence of protein the 
> surface tension of the sample became low enough to leak through the 
> defects of the centeriece
> 
> On Fri, Jun 12, 2009 at 4:51 PM, sumit goswami <ikant11 at gmail.com 
> <mailto: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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-- 
Dr Andrew Leech                   *  Laboratory Head
Technology Facility               *  Molecular Interactions Laboratory
Department of Biology (Area 15)   *  Tel   : +44 (0)1904 328723
University of York                *  Fax   : +44 (0)1904 328804
PO Box 373,  York  YO10 5YW       *  Email : apl3 at york.ac.uk