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Greetings All:<br><br>
What is the experience of others for the stray light level at 250
nm? I know that 3 is the specification, but Is 1.5 abs pushing the
limit at 250 nm? <br><br>
Borries' point about gradients is good, especially given the solubility
issues. A check of the buffer might help.<br><br>
Glen<br><br>
At 10:50 PM 9/6/2009, Paul Leonard wrote:<br>
<blockquote type=cite class=cite cite="">Dear Borries Demeler,<br><br>
Thank-you for replying so quickly.<br><br>
In response to your suggestions I have a few comments (although
these<br>
responses are not directed directly to you but to the RASMB mailing
list<br>
as a whole - please enjoy the labor day weekend rather than responding
to<br>
me).<br><br>
I collected abs measurements at both 280nm and 250nm. For the
250nm<br>
curves there was no problem with the absorbance going above 1.5 abs
units<br>
so I used the same radial range as I used with the interference
optics.<br><br>
I believe this rules out aggregation as being responsible for the<br>
difference in curvature between abs and interference.<br><br>
I really do not know how BeF3- would behave in the experiment.
Actually I<br>
should clarify this a little. BeF3- is actually the addition of 5.3
mM<br>
BeCl2 and 33 mM NaF. Previous studies by NMR have found that at
these<br>
concentrations the major beryllium fluoride species present is BeF3-
(and<br>
not BeF2, BeF42- etc). However BeF2 is relatively insoluble as is
MgF2<br>
which would also be present in small amounts in my sample.<br><br>
However I guess I over complicated the issue (or at least me
greatest<br>
concern) by mentioning the BeF3- experiments. Even in the
experiments<br>
where BeF3- was not present I still do not get the same answer when<br>
fitting the absorbance and interference optics so there must be
something<br>
fundamentally wrong with what I am doing as the two techniques are
viewing<br>
the exact same protein distribution.<br><br>
<br>
kind regards<br><br>
Paul<br><br>
<br><br>
> Paul,<br>
><br>
> A couple of thoughts: You may have some aggregation in your
equilibrium<br>
> experiment (perhaps because of the longer run time). Perhaps in
the<br>
> absorbance<br>
> experiment you are clipping the higher OD's because they are out
of<br>
> the dynamic range of the optics, but in the interference
experiment<br>
> the dynamic range is larger, and you are including the portion of
the<br>
> scan that correlates most with the aggregates.<br>
><br>
> Another possibility is the presence of time invariant noise, but
that<br>
> should have been subtracted from the data by your baseline
scan.<br>
><br>
> Finally, I wonder if the BeF3 refractive index is properly handled
by<br>
> having a meniscus-matched BeF3 subtracted. I don't know the BeF3
density<br>
> off hand, but dependening on density it could for a gradient
and<br>
> significantly contribute to the refractive index signal.<br>
><br>
> Others may have addtl. ideas...<br>
><br>
> -borries<br>
><br>
>> Dear all,<br>
>><br>
>> I have been running both sedimentation velocity and
sedimentation<br>
>> equilibrium AUC experiments on a relatively simple protein
system.<br>
>><br>
>> The sedimentation velocity experiments at multiple protein<br>
>> concentrations<br>
>> show that the protein is just a monomer unless BeF3- is present
(to<br>
>> mimic<br>
>> phosphorylation) and then all of the protein becomes
dimeric.<br>
>><br>
>> However if I run sedimentation equilibrium experiments at
multiple<br>
>> speeds<br>
>> I find that if I use the absorbance optics it is in agreement
with the<br>
>> sedimentation velocity result - protein is just a monomer in the
absence<br>
>> of BeF3- but fits as a dimer if BeF3- is present.<br>
>><br>
>> However if I look at the interference scans for the same cells
(for<br>
>> sedimentation equilibrium AUC) the protein looks considerably
bigger -<br>
>> i.e. it would appear that it is in equilibrium between monomer
and dimer<br>
>> in the absence of BeF3-<br>
>><br>
>> I have also record analytical size exclusion chromatography
profiles for<br>
>> concentrations of the protein all the way from 10uM upto 500 uM
and if<br>
>> BeF3- is absent I cannot see any evidence of dimer
formation. If I add<br>
>> BeF3- the protein is all dimer.<br>
>><br>
>> Therefore I am pretty certain that I should only be seeing
monomer in<br>
>> the<br>
>> absence of BeF3- and dimer if BeF3- is present so I'd like to
know why<br>
>> my<br>
>> interference data makes the protein look larger than it
should. Is<br>
>> there<br>
>> some correction that I need to apply. I have recorded
water blanks at<br>
>> each speed and it makes virtually no difference if I subtract
these<br>
>> scans<br>
>> (Not enough of a correction to account for the additional
curvature in<br>
>> the<br>
>> scans).<br>
>><br>
>> Could it be that the radial calibration is wrong? Is
something wrong<br>
>> with<br>
>> the interference optics? Any explanation would be greatly
appreciated.<br>
>><br>
>> I fear that if we cannot get reliable results for this simple
case using<br>
>> this equipment for more complex systems will become impossible
(at least<br>
>> for sedimentation equilibrium experiments).<br>
>><br>
>> Thank-you for any help you can provide.<br>
>><br>
>> Paul<br>
><br><br>
<br>
Department of Biochemistry<br>
UMDNJ-Robert Wood Johnson Medical School<br>
Center for Advanced Biotechnology and Medicine<br>
679 Hoes Lane<br>
Piscataway, New Jersey 08854-5627<br><br>
Phone: (732) 235-4206<br>
Email: leonard@cabm.rutgers.edu<br><br>
_______________________________________________<br>
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<br>
Glen Ramsay, Ph.D.<br>
Chief Scientist<br>
Aviv Biomedical, Inc.<br>
750 Vassar Avenue<br>
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(732) 370-1300, Ext. 25<br>
(732) 370-1303, FAX<br>
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www.avivbiomedical.com<br>
</a> Skype: GlenAtAviv<br><br>
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