[RASMB] a hiccup in radial space

[John Philo]

Chad,

I think most likely you have a problem with the motion of the slit assembly.
If there is dirt underneath or a metal burr and it moves up and down because
of that you can get glitches and shifts in the OD readings. 

Regarding analyzing the data, if you have a scan at a wavelength where there
should be no absorbance (maybe 350 nm) that might work as a baseline you
could subtract, but on the other hand the size of the glitch might not be
wavelength-independent.

John

-----Original Message-----
From: rasmb-bounces at rasmb.bbri.org [mailto:rasmb-bounces at rasmb.bbri.org] On
Behalf Of Chad Brautigam
Sent: Thursday, December 18, 2008 10:06 AM
To: rasmb at rasmb.bbri.org
Subject: [RASMB] a hiccup in radial space

Dear All,

Whilst checking the data from the first speed of a sedimentation equilibrium
run, I noticed a troubling discontinuity in the data.  It appears in all
three cells, and it is not exactly subtle.  The jump in the data can be as
much as 0.1 OD, and it always occurs betwixt 7.05  
and 7.06 in radial space.  The anomaly occurs at both 280 and 250 nm.   
I attach a graph of three successive scans from the same cell, spaced four
hours apart, for your delectation.  The data were collected in stepping
mode, with 20 replicates.

While I suspect that the heavily glycosylated protein in these samples might
behave nonideally, I suspect that the real problem here is instrumentation,
maybe a sticky rail.  But, before calling my service rep, I thought I'd tap
the collective wisdom of the AUC hive mind.

And a further question:  do y'all think that these data can still be  
analyzed if I simply exclude the affected region from the analysis?   
I'm running in the 6-hole centerpieces, so that means I'd be eliminating
about half of the data from the outer holes.  Alas.

Thanks for any insight you can provide.

Best,
Chad




======================================
Chad A. Brautigam, Ph.D.
Assistant Professor
Department of Biochemistry
The University of Texas
Southwestern Medical Center at Dallas
Department of Biochemistry
5323 Harry Hines Blvd.
Rm. ND10.214G
Dallas, TX 75390-8816
Office:  214-645-6384
Fax:  214-645-6353
Email:  chad.brautigam at utsouthwestern.edu