[RASMB] sedimentation with P20

[John Philo]

John, you should not have any problems running SV on this sample using
absorbance detection. The tween 20 micelles sediment near 2 S (slow for
their MW because the detergent density is less than for protein). 

For reasons too complex to go into here, it is quite difficult to match the
Tween signals in the sample and reference channels. If the Tween is
reasonably pure you probably won't see more than a few hundredths of an OD
from the UV-absorbing impurities in it (pure Tween does not absorb at 280
nm). Thus I would suggest simply leaving the Tween out of the reference
channel, and then also running a control of Tween-containing buffer versus
water so you know exactly how fast the micelles sediment.

John

From: RASMB [mailto:rasmb-bounces at list.rasmb.org] On Behalf Of John Sumida
Sent: Friday, August 05, 2016 1:00 PM
To: RASMB <rasmb at list.rasmb.org>
Subject: [RASMB] sedimentation with P20

 

Dear RASMB,

 

I would like to design an experiment that would confirm (or not) the
presence of a monomer with an expected mass of 60kDa.  This protein is being
prepped for an SPR experiment but has been shown to form significant
aggregates in the absence of tween 20.  We would like to confirm that in the
presence of 0.05% Tween, the solution is primarily monomeric.

 

DLS analysis of just the buffer containing 0.05%Tween shows a peak at 3nm.
Analysis of the protein in the same buffer, likewise indicates a peak at
3nm.

 

Calculating the stokes radius from the theoretical diffusion value (8.15
Ficks), the expected stokes radius of the protein is 2.4nm.

 

Thus the problem is how to distinguish between protein and micelle which
appear to have similar stokes radii.

 

SEC is one approach that we are trying, but in thinking about getting to an
answer with another method to bolster the SEC data, my understanding is that
if there were no thermodynamic non-ideality present, an equilibrium
measurement monitoring the absorbance of the protein may be another viable
approach since one would not have to account for any hydrodynamic
non-ideality that I expect would arise in a transport (Sed Vel) measurement.

 

I would be interested in your thoughts and comments, and as always I thank
you for your time and kind consideration.

 

Best regards,

John Sumida Ph.D.

Bionalytical Core Facility Manager

University of Washington

Molecular Engineering & Sciences Rm G22

 

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