[RASMB] Photomultiplier tube disturbances?

Troy Sorensen ts1172 at rams.colostate.edu
Tue Oct 12 13:16:22 PDT 2010


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
-------------- next part --------------
A non-text attachment was scrubbed...
Name: 101010_0.75_intensity conversion.png
Type: image/png
Size: 307434 bytes
Desc: not available
URL: <http://list.rasmb.org/pipermail/rasmb-rasmb.org/attachments/20101012/c7d378e9/attachment.png>
-------------- next part --------------
A non-text attachment was scrubbed...
Name: 101010_0.75_zoomed.png
Type: image/png
Size: 227407 bytes
Desc: not available
URL: <http://list.rasmb.org/pipermail/rasmb-rasmb.org/attachments/20101012/c7d378e9/attachment-0001.png>


More information about the RASMB mailing list