[RASMB] XL-I poor data troubleshooting

[Leech, AP]

Hi Mark,

I wouldn't worry about the curious figures from the S values - I think
this arises from the way SEDFIT spaces the S values over the range you
have specified (it uses whatever number of values you chose for the
resolution parameter, default 50).

I would go back to basics:

1. Can you see a problem in the interference optics too?
2. Runs some scans of empty holes, empty cells and the counterbalance
itself, using the intensity mode to see if your optics are getting
reasonable amounts of light through the system (~thousands of counts,
more or less flat; a wavelength scan to check the wavelength accuracy
might be a good idea too). The counterbalance holes should stop letting
light through at 5.85 cm and open up again at 7.15. If that's all ok,
check some samples the same way.
3. Try another program if it may be that you are doing something
unaccountably wrong with SEDFIT. Does it give any of the explanatory
error messages for your data?
4. Compare scans previous to the problem with recent ones to see if
there is any obvious change. Post some up for us to look at if it would
help.
5. You don't say anything about your samples, but lysozyme is quite
small - I'd use BSA for a standard (s~4.2 with usually a touch of dimer
about 6.3). Is there any other corroborating data about the samples
(e.g. gel filtration runs)?

Hope this is useful,

Andrew

Mark Agacan wrote:
> Hello,
> 
> I am having problems with the data being collected on my XL-I.  For all the samples I have studied lately, the rmsd has been 0.2 - 0.8, the residual bit maps show large contrast from black to white running in a diagonal across the whole data range, and the MW values output from SedFit bear no resemblance to what I have been  expecting.    
> 
> I have also noticed that the S-values just don't seem right:  e.g. cell 2 = 6.545455; cell 4 = 7.272728; cell 6 = 8.383839;
> The repetition in the numbers after the decimal point are surely artefactual?  I've never seen a dataset consistently produce numbers like this.
> 
> I have used different windows and centerpieces in an attempt to get better data but to no avail.  My sample preparation is standard and the samples and buffers are standard, e.g.  50 mM Tris 100 mM NaCl.  All samples show up as distinct bands on gels with MW values as expected.
> 
> I recently had the radial servo replaced on my XL-I - could this or malfunctioning / poorly calibrated optics be to blame?  I will run a lysozyme sample (MW = 14.4 kDa) in the next few days in place of a suitable standard.  The residual bitmap and high rmsd makes me think that the optics may not be properly aligned and that they are imaging part of the walls of the centerpiece, instead of the sample.
> 
> It's not impossible that the samples actually really do have the mass and S-values as output, but I am highly suspicious, and the rmsd values are terrible.
> 
> Has anyone had issues such as this, and if so have you any recommendations or ideas that I can try in order to get to the bottom of this problem?
> 
> Best Regards,
> 
> Mark
> 
> 
> _____________________________________
> Dr Mark Agacan
> Scientific Officer,
> Division of Biological Chemistry 
> and Drug Discovery,
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> University of Dundee,
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-- 
Dr Andrew Leech                   *  Laboratory Manager
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