<html>
<head>
<style><!--
.hmmessage P
{
margin:0px;
padding:0px
}
body.hmmessage
{
font-size: 10pt;
font-family:Tahoma
}
--></style>
</head>
<body class='hmmessage'>
Have all scans been aquired at the same wavelength? Sometimes it jumps between scans by few nm.<BR>
Best<BR>
Titus<BR> <BR>> From: jphilo@mailway.com<BR>> To: rasmb@server1.bbri.org<BR>> Date: Tue, 12 Oct 2010 16:33:50 -0700<BR>> Subject: Re: [RASMB] Photomultiplier tube disturbances?<BR>> <BR>> Troy, have you read the past RASMB discussions of problems arising from<BR>> trying to run samples in the reference channel? Remember that the instrument<BR>> is designed to scale the photomultiplier voltage and the programmable gain<BR>> amplifier based on the intensity it sees at 6.5 cm before it starts each<BR>> radial scan. <BR>> <BR>> It seems to me you are seeing exactly what has been discussed previously:<BR>> when the DNA boundary in the reference channel sediments to near 6.5 cm, the<BR>> instrument is readjusting the PMT voltage and/or the PGA gain, and this<BR>> produces a break in the pseudo-absorbance data for both channels of that<BR>> cell. Basically pseudo-absorbance only works correctly if the instrument<BR>> ends up not changing the PMT voltage or PGA setting for that cell at all<BR>> during the run; if the instrument decides it needs to do so, either because<BR>> something is sedimenting in the reference, or just because the lamp<BR>> intensity is drifting for some reason, you are screwed.<BR>> <BR>> Whenever you run in pseudo-absorbance mode you are asking the instrument to<BR>> do something it wasn't ever intended to do. When the OD in the reference<BR>> channel is quite low it usually works okay, but you are risking getting low<BR>> quality or un-interpretable data. You also put yourself in a position where<BR>> you can't complain to Beckman that something is wrong.<BR>> <BR>> I don't know if Tom Laue's AOS operating software can get into the machine<BR>> internals and modify this re-scaling behavior. I tend to doubt it, but if it<BR>> could that would help tremendously when running in pseudo-absorbance mode.<BR>> <BR>> As Borries said, probably the only way to get around this issue is to lower<BR>> the OD in the reference channel, or just run in normal absorbance mode with<BR>> buffer in the reference.<BR>> <BR>> John<BR>> <BR>> -----Original Message-----<BR>> From: rasmb-bounces@rasmb.bbri.org [mailto:rasmb-bounces@rasmb.bbri.org] On<BR>> Behalf Of Troy Sorensen<BR>> Sent: Tuesday, October 12, 2010 1:16 PM<BR>> To: rasmb@rasmb.bbri.org<BR>> Subject: [RASMB] Photomultiplier tube disturbances?<BR>> <BR>> Hello,<BR>> <BR>> I am performing sedimentation velocity experiments in a XL-I in intensity<BR>> mode, scanning at 260 nm. My samples are a protein-DNA nucleosomal array<BR>> complex. I am running a four hole rotor with cell 1 sector 1 as my ddH20<BR>> blank, the remaining 5 sectors contain a DNA concentration equilavent to<BR>> ~0.2 absorbance units. I am spinning the rotor at 20K rpms for about 2.5<BR>> hours. The intensity of the machine at 259 nm is relatively high at ~45000<BR>> units.<BR>> <BR>> My issue seems to be with the photomultiplier displacing one or more scans<BR>> (typically a decrease in magnitude) during the course of the run. I do not<BR>> believe this is from sample sedimenting as the decrease in magnitude is the<BR>> same and at the same scan number for both sectors in a cell, but at<BR>> different scan numbers for different cells. The downward shift doesn't<BR>> always happen for each cell either. It may occur in just 1, 2, or all 3<BR>> cells. I can have a run where it only occurs in one cell and the other two<BR>> cells are fine.<BR>> <BR>> The images attached are from the Ultrascan software that converts the<BR>> intensity data to pseudo-absorbance, and a zoomed in visual of the<BR>> psuedo-absorbance data. I would be more than happy to provide more<BR>> information if my inquiry is to vague!<BR>> <BR>> If this is due to the high intensity at 259 nm, would using scanning at a<BR>> wavelength like 262 nm (~22000 units) circumvent this problem?<BR>> Any other suggests, comments, concerns would be greatly appreciated.<BR>> <BR>> Thanks for your time,<BR>> Troy Sorensen<BR>> <BR>> Hansen Lab<BR>> Department of Biochemistry and Molecular Biology Colorado State University,<BR>> Fort Collins CO<BR>> <BR>> _______________________________________________<BR>> RASMB mailing list<BR>> RASMB@rasmb.bbri.org<BR>> http://rasmb.bbri.org/cgi-bin/mailman/listinfo/rasmb<BR>> <BR>> <BR> </body>
</html>