[RASMB] Speed of Interference Optics vs. Absorbance Optics

[Borries Demeler]

> 
> Folks,
> 
> I have a sample, a membrane protein with 0.01% DDM. I collected data at
> 45000 rpm, 4 min/scan. The sample is very heterogeneous but I am getting a
> decent peak from the absorbance data (280 nm) at 7.4 S, but the interference
> data gives me a peak at 6.5 S. I am trying to calculate the amount of
> detergent bound so I need to align the interference data with absorbance
> data. This is the first time in which I have gotten the peaks from the
> interference data not lining up with absorbance data. Is this due to the
> time it takes to make an absorbance scan relative to the interference scans?
> And/or I just spun the sample too hard? Or is the DDM affecting the
> sedimentation? See the attached powerpoint. 
> 
> Yes, the slit assembly is functioning properly J   
> 
> Any suggestions will be helpful! 
> 
> Best,
> 
> Lake
> 
>  
> 
> PS I collected data during the same run with the same sample except it had
> GFP attached. The interference peak shifted accordingly. The DDM peak lined
> up well also. 
> 

Dear Lake,

the reason you see different s-value distributions is most likely
dependent on the fact that you are looking at different properties with
the 2 optical systems. The refractive indices of protein and detergent
are quite different from their absorbances at 280 nm. Since their
sedimentation/diffusion are likely different as well, I would not expect
their sedimentation patterns to coincide between the two experimental
systems. Once you start averaging signals from different species into
s-distributions, it is not unexpected that these distributions will
shift when switching optical systems. If you know exactly the RI and
absorbances of each species, you may be able to figure out how much
of each is free and how much is bound.

The slide with the sed. coeff. distribution would not load correctly, 
so I cannot see your results.

Good luck, -Borries