[RASMB] systematic differences

[Leech, AP]

Dear all,

A quick back of the envelope calculation assuming the width of a sector
is 3mm, makes Jack's 0.05 cm discrepancy equal to 18 mm^3 (18 uL) difference
in volume in the cell (for about 420 uL filling), which would correspond
to a difference in width of ~0.125 mm along the solution column. Does
anyone know if this is within manufacturing tolerance of the cells? It
seems a little large. Perhaps the CAMIS people have an idea of the spread
of cell measurements?

I presume Jack tracked the discrepancy by not refilling the cells but
shaking them up then moving them?

Andrew


John Philo wrote:
> Jack, since I haven't seen any other responses to your message from last
> week I will jump in and offer one theory.
> 
> You haven't said how you are filling these cells so reproducibly that you
> can measure this correlation between meniscus position and cell number. You
> also haven't said what type of centerpieces you are using, but I will guess
> they are charcoal epon. Then if that correlation is correct it means the
> channel volumes are different for different centerpieces (the same volume
> gives a different column height). That I think can only be true if the
> center rib is bowed, probably due to a leak at run speed (one channel empty,
> the other full). That is, I think this whole exercise is telling you that at
> least two of your centerpieces are damaged.
> 
> Also, regarding your "correction factor", how can you say which cell gives
> the "right" sedimentation coefficient and which ones need correction?
> 
> John
> 
> -----Original Message-----
> From: rasmb-bounces at rasmb.bbri.org [mailto:rasmb-bounces at rasmb.bbri.org] On
> Behalf Of Jack Kornblatt
> Sent: Thursday, August 31, 2006 12:28 PM
> To: rasmb at server1.bbri.org
> Subject: [RASMB] systematic differences
> 
> 
> This note relates to systematic between cells in the AUC.
> 
> The problem, as I mentionned in my note, is that there is a difference in
> the determined s values of the same protein in different cells of AUC. This
> problem has apparently been seen by others as well. The question is: what is
> the cause of the problem? There is no firm answer. There is good
> speculation.
> 
> First, I would like to thank you all for your comments. I have followed your
> advice. Cell 1 went into rotor position 2 and cell 2 went into rotor
> position 1. The bias that was found when the cells were in their normal
> positions (cell 1 in rotor position 1, etc) followed the cells; the data are
> clean and clear. In addition to the systematic difference between the s
> values that I find, there is a systematic difference in the position of the
> menisci (meniscuses). Cell 1 which uniformly gives an s value higher by 0.6%
> (from
> stats) has a meniscus that is uniformly closer to the center of the rotor by
> about .05 cm. My gut feeling is that this should not in any way influence
> the values of s that we determine so long as the meniscus is constant
> throughout the run. Once I leave the gut, I'm hard pressed to find an
> explanation for why the value of s should vary. I now apply a correction
> factor to the s values obtained in each cell.
>  
> Last point from a biologist. "I'm learning far more about analytical
> ultracentrifugation than I ever thought I would want to."
> 
> All the best and thanks once more
> 
> jack
> jack a kornblatt
> 
> 
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-- 
Dr Andrew Leech                   *  Head of the Molecular
Technology Facility               *   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