[RASMB] Photomultiplier tube disturbances?

[Tom Laue]

  Hi Troy-
I think John has the problem identified. I wish AOS could change the 
gains, but they are set by the XLA, and inaccessible the external computer.
Best wishes,
Tom

On 10/12/2010 7:33 PM, John Philo wrote:
> 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
>
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-- 
Department of Biochemistry and Molecular Biology
University of New Hampshire
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