[RASMB] Sedimentation coefficient of human serum albumin monomer and good protein standard with

Tue Dec 18 12:01:33 PST 2007

I have used the Stafford method twice, 10 years apart and got great data that agreed with 0.1 C from 4 to 40oC.  And the values were close to set points; at 25 it measured 24.7, for example.  Yes there is low intensity at 600 nm, but I suspect the noise is because you forgot to unflip the 400 nm cut off filter, Jack!  You get the best results if you integrate the entire spectra from 400 to 750 nm and compare the integrated XLA with the integrated Cary data at each temp, corrected for path length of course.
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Dr. John J. "Jack" Correia
Department of Biochemistry
University of Mississippi Medical Center
2500 North State Street
Jackson, MS  39216
(601) 984-1522                                 fax (601) 984-1501                             email address: jcorreia at biochem.umsmed.edu     homepage location: http://biochemistry.umc.edu/correia.html dept homepage location:    http://biochemistry.umc.edu/ -------------------------------------------------------------------

>>> On 12/18/2007 at 11:29 AM, in message <47680380.1090209 at alcor.concordia.ca>, Jack Kornblatt <krnbltt at alcor.concordia.ca> 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
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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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