[RASMB] Low s value peaks

John Philo jphilo at mailway.com
Fri Jan 23 14:54:00 PST 2004


Samantha,
 
It is very common with the c(s) method to see what you are calling a
'half-peak' at the left end, but this is often a 'false' peak. Basically it
arises because the data do not go out to long enough times to provide any
real information about the very small sedimentation coefficients, because
they never pull significantly away from the meniscus. If you are analyzing
data taken at 30K rpm, you probably really can't 'see' any species below 0.5
S (if that). If you allow the fitter to play with parameters that have no
real influence on the data, it is virtually guaranteed that it will come up
with some nonsense trying to fit what is really noise in the data.
 
So you probably want to start the distribution at a higher s value, but
doing that doesn't always get rid of your half-peak. Once you realize it is
false, just ignore it. If you need a graph with that peak gone, just copy
and paste the distribution table into another graphing program and delete
those low sedimentation coefficient points.
 
It is of course possible with interference data that you are actually
detecting some sedimentation of buffer components, but that is much less
likely at 30K. As Walt Stafford often points out, even if you use a
dialysate the buffer sedimentation will not perfectly cancel unless the
reference meniscus is matched to the sample meniscus.
 
John Philo

-----Original Message-----
From: rasmb-admin at rasmb-email.bbri.org
[mailto:rasmb-admin at rasmb-email.bbri.org] On Behalf Of Jones, Samantha
Sent: Friday, January 23, 2004 9:20 AM
To: rasmb at rasmb-email.bbri.org
Subject: [RASMB] Low s value peaks


Hello all
I have been trying to analyse some interference data on Sedfit and have been
repeatedly seeing a large 'half' peak at very low s values on my s value
distributions.
On the continuous c(s) distributions after fitting to range of 0.1s to 10s
the distribution line starts at c(s) values of 1.5 to 0.5 on 0 on the x
axis, dropping to 0 on the y axis by 0.1s value. This is still the case
after changing the parameters to 0.01s to 10s. 
The buffer used at first was 10mM NaAcetate, 0.2% Azide and 2mM DTT pH 4. At
first I thought it could be the DTT but I saw the same thing using a sample
in a buffer 10mM Tris 10mM Na Acetate, 0.2% Azide no DTT pH 8. 
I have seen this spinning at 30K and 50K for about 16hours.
 
Could Azide be the problem? I don't expect either sample to be contaminated,
however anything of this size would have been missed by the SDS-PAGE gel run
on both samples.
 
Thank you
 
Samantha Jones
Samantha Jones 
MRC Prion Unit 
St Marys Hospital 
Norfolk Place 
London 
W2 1PG 

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