[RASMB] systematic differences

[John Philo]

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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