[RASMB] buffer components and IF optics

[Peter Schuck]

John and Samantha,

I think your questions about the use of buffer components in conjunction 
with the IF optics are related, as far as sedimentation velocity 
experiments are concerned.

Regarding the use of IF optics in the presence of detergents, there is the 
problem that you see the detergent sediment or float, whereas it is 
frequently not visible in the absorption optics.  I don't think that seeing 
the detergent is necessarily a problem in IF. It can be a problem in UV 
because of the limited dynamic range of absorption, but in IF, it is just a 
signal from an extra component superimposed to your signal from the 
sedimenting protein.  I've done experiments with Triton and LDAO, and in 
both cases it was possible to describe the detergent signal with a very 
high degree of precision by a single component Lamm equation 
solution.  (The sedimentation or flotation parameters can be measured 
separately.)  You can take this superimposed species easily into account in 
the data analysis (in SEDFIT, there are appropriate models for ls-g*(s) and 
c(s)).  In the end, with regard to sedimentation velocity experiments, I 
think it may actually be an advantage to see what the detergent does.

Regarding high concentrations of DTT, I have no hands-on experience with 
this situation in particular, but if it's not possible to precisely match 
the sample and reference sectors in the buffer composition, you'll see the 
sedimentation of DTT.  Also, I'm not sure if the refractive index increment 
of DTT could possibly change with oxidation state, and in this way create a 
mismatch between the two sectors.  But again, it should be no problem to 
take this into account in the data analysis (assuming that you have an 
excess signal from DTT in the sample sector), by treating this as an extra 
sedimenting species.

I've run quite frequently into problems like that, where collaborators gave 
me samples that were not properly matched to the reference buffer.  Excess 
NaCl, for example, can be very reproducibly modeled as an extra sedimenting 
low-Mw component without noticeable degradation of the information of the 
high-Mw sedimentation (the sedimentation patterns just look 
different).  However, a drawback is that the bottom position will have to 
be optimized during the data analysis, because of the back-diffusion of the 
extra small Mw species.

Peter