[RASMB] Photomultiplier tube disturbances?

[John Philo]

Troy, have you read the past RASMB discussions of problems arising from
trying to run samples in the reference channel? Remember that the instrument
is designed to scale the photomultiplier voltage and the programmable gain
amplifier based on the intensity it sees at 6.5 cm before it starts each
radial scan. 

It seems to me you are seeing exactly what has been discussed previously:
when the DNA boundary in the reference channel sediments to near 6.5 cm, the
instrument is readjusting the PMT voltage and/or the PGA gain, and this
produces a break in the pseudo-absorbance data for both channels of that
cell. Basically pseudo-absorbance only works correctly if the instrument
ends up not changing the PMT voltage or PGA setting for that cell at all
during the run; if the instrument decides it needs to do so, either because
something is sedimenting in the reference, or just because the lamp
intensity is drifting for some reason, you are screwed.

Whenever you run in pseudo-absorbance mode you are asking the instrument to
do something it wasn't ever intended to do. When the OD in the reference
channel is quite low it usually works okay, but you are risking getting low
quality or un-interpretable data. You also put yourself in a position where
you can't complain to Beckman that something is wrong.

I don't know if Tom Laue's AOS operating software can get into the machine
internals and modify this re-scaling behavior. I tend to doubt it, but if it
could that would help tremendously when running in pseudo-absorbance mode.

As Borries said, probably the only way to get around this issue is to lower
the OD in the reference channel, or just run in normal absorbance mode with
buffer in the reference.

John

-----Original Message-----
From: rasmb-bounces at rasmb.bbri.org [mailto:rasmb-bounces at rasmb.bbri.org] On
Behalf Of Troy Sorensen
Sent: Tuesday, October 12, 2010 1:16 PM
To: rasmb at rasmb.bbri.org
Subject: [RASMB] Photomultiplier tube disturbances?

Hello,

I am performing sedimentation velocity experiments in a XL-I in intensity
mode, scanning at 260 nm. My samples are a protein-DNA nucleosomal array
complex.  I am running a four hole rotor with cell 1 sector 1 as my ddH20
blank, the remaining 5 sectors contain a DNA concentration equilavent to
~0.2 absorbance units.  I am spinning the rotor at 20K rpms for about 2.5
hours.  The intensity of the machine at 259 nm is relatively high at ~45000
units.

My issue seems to be with the photomultiplier displacing one or more scans
(typically a decrease in magnitude) during the course of the run.  I do not
believe this is from sample sedimenting as the decrease in magnitude is the
same and at the same scan number for both sectors in a cell, but at
different scan numbers for different cells.  The downward shift doesn't
always happen for each cell either.  It may occur in just 1, 2, or all 3
cells.  I can have a run where it only occurs in one cell and the other two
cells are fine.

The images attached are from the Ultrascan software that converts the
intensity data to pseudo-absorbance, and a zoomed in visual of the
psuedo-absorbance data. I would be more than happy to provide more
information if my inquiry is to vague!

If this is due to the high intensity at 259 nm, would using scanning at a
wavelength like 262 nm (~22000 units) circumvent this problem?
Any other suggests, comments, concerns would be greatly appreciated.

Thanks for your time,
Troy Sorensen

Hansen Lab
Department of Biochemistry and Molecular Biology Colorado State University,
Fort Collins CO