[RASMB] What is wrong with my XLA?

John Philo jphilo at mailway.com
Sun Jul 18 20:25:17 PDT 2010


Steve, I agree with Borries, I think this may be a problem that is related
to trying to run a second sample in the reference channel. If I were you I
would try running some control sample in standard absorbance mode and see
whether that behaves normally.

John 

-----Original Message-----
From: rasmb-bounces at rasmb.bbri.org [mailto:rasmb-bounces at rasmb.bbri.org] On
Behalf Of Borries Demeler
Sent: Friday, July 16, 2010 11:29 PM
To: smcbryan
Cc: rasmb at server1.bbri.org
Subject: Re: [RASMB] What is wrong with my XLA?

> 
> Fellow centrifugationistas,
> Attached find two .png files of 1) two sample meniscus regions from 
> data collected in the intensity mode at 280nm, and 2) the resulting 
> data from one of these samples after time independent noise subtraction.
> almost certainly there is a problem with my slit assembly, resulting 
> in the menisci which defy fitting. My question is, is the very 
> anomalous data (which does not fit via van Holde-Weischet analysis)a 
> result of ONLY the meniscus jumping about, or is there perhaps another 
> cause superimposed on the slit assembly?
> Finally, there is a noise signature at 6.35cm in all sectors of all 
> cells always with this machine. I have cleaned the glass filter in the 
> bottom of the rotor chamber that protects the lamp, and blown some 
> compressed air gently into the slit in the monochrometer assembly. 
> Neither act has removed the offending noise. Any further suggestions for
removal are appreciated.
> cheers,
> steve

Steve,

I am with John on the meniscus issue - can you plot the first, middle and
last scan in three different colors, it would be nice to see if there is a
pattern to the meniscus drift.

Secondly, the scans in the velocity scan seem to jump around and apparently
seem to have different scalings. Assuming you collected scans from both
reference and sample channel, are you sure that the absorbance of the sample
plus baseline absorbance in the reference cell did not exceed 0.5 OD? It
almost looks like it rescaled on you. If not that, perhaps it was acquired
at different wavelengths. The scans you show do not show a regular velocity
pattern. Something is screwed up either with your expt.  design or your
instrument, there is not enough information to determine which.  You should
run some diagnostics on your machine and call Beckman service for assistance
- it is not clear what is going on here, but too high a concentration in the
reference channel will cause a reset of the gain settings for the PM tube,
and you want to avoid this. Even if your sample OD is 0.3 or less, if you
have baseline absorbance from buffer or reductants, this can seriously mess
you up. I just assume you are not running DTT in your sample, correct?

-b.
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