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

[walter stafford]

Jack et al.

You must integrate the spectrum between the wavelengths indicated in the 
paper in order to get sufficient precision. A simple OD measurement at 
one wavelength will definitely not do. If you do that carefully, and 
with a sufficient number of replicates, it works as advertised. In fact 
it is the low sensitivity in the red region that led us to resort to 
integration.

Walter

Jack Kornblatt wrote:
> 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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