[RASMB] Sedimentation coefficient of human serum albumin monomer and good protein standard with well established s value

[Jack Kornblatt]

Hello all
I would like to add a note of caution to Arthur's suggestion. I tried  
using Walter's approach to determining the temperature of the rotor 
during the run. The cobalt solutions gave beautiful data in my old Cary 
but gave "rediculous" data in the centrifuge. When  I scanned the cobalt 
solution while the centrifuge was running, it was clear that the machine 
did not have a red sensitive PM. The accuracy and precision of Walter's 
approach is  dependent on measuring an OD in the red. If that OD is in 
error, kiss goodbye to good data.
best regards to all
jack kornblatt

Arthur Rowe wrote:

>     Greetings, everyone
>
>     This issue is an obvious signal for a old hobby-horse of mine to
>     have a run out in the field.
>
>     The concept of a standard s value for some protein (or whatever)
>     is a lovely idea. But two things are involved: one of them is
>     getting suitable standard solute(s), which may be possible, say
>     RNAse as one idea; but the the other thing is getting all the
>     parameters to be either known or measurable. I have discussed in
>     some detail all the factors which limit the precision and the
>     accuracy of s values (Errington & Rowe 2003). The most serious
>     matter by a way is the rotor temperature, and this is not known in
>     the XL-I/A to the precision needed to take advantage of the
>     precision inherent in the definition of s values by SV analysis.
>
>     However, if you are prepared, or someone is, to check out the
>     absolute temperature of a rotor using Walter Stafford's
>     colorimetric method, then it just needs one definition of the
>     'real' s values for one or two well known, stable and monomeric
>     proteins^ for everyone else to be able to benefit. Volunteers?
>
>     Seasonal wishes to all
>
>     Arthur
>
>     ^or something else, such as small colloidal gold spheres, from a
>     batch available to all. They give beautiful SV diagrams. Or
>     apoferritin - always has some dimers present, but they are
>     resolved away, so who cares?
>
> --
> *******************************************************
> Arthur J Rowe
> Professor of Biomolecular Technology
> NCMH Business Centre
> University of Nottingham
> School of Biosciences
> Sutton Bonington
> Leicestershire LE12 5RD   UK
>
> Tel:        +44 (0)115 951 6156
>            +44 (0)116 271 4502
> Fax:        +44 (0)115 951 6157
> email:      arthur.rowe at nottingham.ac.uk
> Web:        www.nottingham.ac.uk/ncmh/business
> *******************************************************
>
>
>
>     N Errington, A J Rowe (2003)  "Probing conformation and
>     conformational change in proteins is optimally undertaken in
>     relative mode" European Biophysics Journal 32 (5) 511-517
>
>
>
>
>     Hi! Everyone
>     We are looking for some "protein standard" to qualify our
>     sedimentation
>     velocity method. We think that HSA may be a good candidate for the
>     standard. Does anyone know the sedimentation coefficient of human
>     serum
>     albumin monomer (corrected for buffer density and viscosity as well as
>     vbar)? Some literatures said HSA has a sedimentation coefficient
>     of 4.6 s,
>     this is significantly higher than the value of the monomer
>     sedimentation
>     coefficient of HSA I got by analyzing the SV data of Sigma HSA
>     using C(s)
>     model. The C(s) profile of this Sigma HSA showed that the product
>     contains
>     about 80% monomer, 17% dimer and 3% trimer. So I guess the 4.6 s value
>     reported in some literatures is the weight average apparent s
>     value of the
>     HSA monomer, dimer and trimer (or even some oligomers) in the sample
>     instead of the apparent s value of HSA monomer. Therefore it will
>     be very
>     helpful for me if anyone can tell me the accurate s value of HSA
>     monomer.
>     Also the suggestions for better protein standard (better commercial
>     available) with a well established s value will be very helpful.
>
>     Thanks!
>
>     Yiming
>
>
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