[RASMB] Why is my interference data different from teh absorbance

Tom Laue Tom.Laue at unh.edu
Tue Sep 8 05:46:24 PDT 2009


Hi-
You can use the ratio of absorbances (e.g. 280/230) to test for stray 
light, so long as the extinction coefficients are constant. My 
experience is that the upper limit (with my instrument, which is very 
old) at 230 is ~1.2.
The tendency is for the absorbance to be underestimated at high 
concentrations, which would appear as a lower apparent molecular weight 
or a positive second virial coefficient.
Best wishes,
Tom

Glen Ramsay wrote:
> Greetings All:
>
> 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?
>
> Borries' point about gradients is good, especially given the 
> solubility issues.  A check of the buffer might help.
>
> Glen
>
> At 10:50 PM 9/6/2009, Paul Leonard wrote:
>> Dear Borries Demeler,
>>
>> Thank-you for replying so quickly.
>>
>> In response to your suggestions I have a few comments (although these
>> responses are not directed directly to you but to the RASMB mailing list
>> as a whole - please enjoy the labor day weekend rather than responding to
>> me).
>>
>> I collected abs measurements at both 280nm and 250nm.  For the 250nm
>> curves there was no problem with the absorbance going above 1.5 abs units
>> so I used the same radial range as I used with the interference optics.
>>
>> I believe this rules out aggregation as being responsible for the
>> difference in curvature between abs and interference.
>>
>> I really do not know how BeF3- would behave in the experiment.  
>> Actually I
>> should clarify this a little.  BeF3- is actually the addition of 5.3 mM
>> BeCl2 and 33 mM NaF.  Previous studies by NMR have found that at these
>> concentrations the major beryllium fluoride species present is BeF3- (and
>> not BeF2, BeF42- etc).  However BeF2 is relatively insoluble as is MgF2
>> which would also be present in small amounts in my sample.
>>
>> However I guess I over complicated the issue (or at least me greatest
>> concern) by mentioning the BeF3- experiments.  Even in the experiments
>> where BeF3- was not present I still do not get the same answer when
>> fitting the absorbance and interference optics so there must be something
>> fundamentally wrong with what I am doing as the two techniques are 
>> viewing
>> the exact same protein distribution.
>>
>>
>> kind regards
>>
>> Paul
>>
>>
>>
>> > Paul,
>> >
>> > A couple of thoughts: You may have some aggregation in your equilibrium
>> > experiment (perhaps because of the longer run time). Perhaps in the
>> > absorbance
>> > experiment you are clipping the higher OD's because they are out of
>> > the dynamic range of the optics, but in the interference experiment
>> > the dynamic range is larger, and you are including the portion of the
>> > scan that correlates most with the aggregates.
>> >
>> > Another possibility is the presence of time invariant noise, but that
>> > should have been subtracted from the data by your baseline scan.
>> >
>> > Finally, I wonder if the BeF3 refractive index is properly handled by
>> > having a meniscus-matched BeF3 subtracted. I don't know the BeF3 
>> density
>> > off hand, but dependening on density it could for a gradient and
>> > significantly contribute to the refractive index signal.
>> >
>> > Others may have addtl. ideas...
>> >
>> > -borries
>> >
>> >> Dear all,
>> >>
>> >> I have been running both sedimentation velocity and sedimentation
>> >> equilibrium AUC experiments on a relatively simple protein system.
>> >>
>> >> The sedimentation velocity experiments at multiple protein
>> >> concentrations
>> >> show that the protein is just a monomer unless BeF3- is present (to
>> >> mimic
>> >> phosphorylation) and then all of the protein becomes dimeric.
>> >>
>> >> However if I run sedimentation equilibrium experiments at multiple
>> >> speeds
>> >> I find that if I use the absorbance optics it is in agreement with the
>> >> sedimentation velocity result - protein is just a monomer in the 
>> absence
>> >> of BeF3- but fits as a dimer if BeF3- is present.
>> >>
>> >> However if I look at the interference scans for the same cells (for
>> >> sedimentation equilibrium AUC) the protein looks considerably bigger -
>> >> i.e. it would appear that it is in equilibrium between monomer and 
>> dimer
>> >> in the absence of BeF3-
>> >>
>> >> I have also record analytical size exclusion chromatography 
>> profiles for
>> >> concentrations of the protein all the way from 10uM upto 500 uM and if
>> >> BeF3- is absent I cannot see any evidence of dimer formation.  If 
>> I add
>> >> BeF3- the protein is all dimer.
>> >>
>> >> Therefore I am pretty certain that I should only be seeing monomer in
>> >> the
>> >> absence of BeF3- and dimer if BeF3- is present so I'd like to know why
>> >> my
>> >> interference data makes the protein look larger than it should.  Is
>> >> there
>> >> some correction that I need to apply.  I have recorded water blanks at
>> >> each speed and it makes virtually no difference if I subtract these
>> >> scans
>> >> (Not enough of a correction to account for the additional curvature in
>> >> the
>> >> scans).
>> >>
>> >> Could it be that the radial calibration is wrong?  Is something wrong
>> >> with
>> >> the interference optics?  Any explanation would be greatly 
>> appreciated.
>> >>
>> >> I fear that if we cannot get reliable results for this simple case 
>> using
>> >> this equipment for more complex systems will become impossible (at 
>> least
>> >> for sedimentation equilibrium experiments).
>> >>
>> >> Thank-you for any help you can provide.
>> >>
>> >> Paul
>> >
>>
>>
>> Department of Biochemistry
>> UMDNJ-Robert Wood Johnson Medical School
>> Center for Advanced Biotechnology and Medicine
>> 679 Hoes Lane
>> Piscataway, New Jersey 08854-5627
>>
>> Phone: (732) 235-4206
>> Email: leonard at cabm.rutgers.edu
>>
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>
>
>    Glen Ramsay, Ph.D.
>    Chief Scientist
>    Aviv Biomedical, Inc.
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-- 
Department of Biochemistry and Molecular Biology
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Phone: 603-862-2459
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