>from the overall lyophilized weight and still it came back high by
20%.
>I'm not sure if extraction with DMF before moisture analysis is able
to
>strip protein bound water off. Do you know?
>
>The counter-ion effect is something we are analyzing. We have
desalted the
>protein into H20 so the contribution of unbound salt is not there.
The Pi
>of the protein I believe is around 5 and the pH of the desalted
material
>into H20 is around 7. We've sent the protein for sodium analysis to
>analyze this counter-ion effect you mentioned.
>
>In you experience, what contribution weight does this counter ion
effect
>have vs. the bound H20 effect? Are we on the right path? Thank you
for
>your help. We will try to "dry" the protein down further.
>
>Best regards, John
John,
The effects of ion binding are very familiar to physical chemists who
do things like trying to interpret the distribution of proteins in
centrifugal fields in terms of molecular weight. In that case, the
difference between bound solvent + ions and bulk solvent can have a
significant effect due to buoyancy. In the case of the extinction
coefficient, the weight of the (assumed) protein may vary depending on
the extent of interaction with solutes, so it's hard to predict which
will have the greater effect. Have you considered doing AAA for weight?
The accuracy for determining the <italic>amount of protein</italic> is
much higher than compositional error. By summing the pmoles of amino
acids you should get to 95%+ accuracy and this is independent of the
presence of bound counterions as it involves no weighing.
regards
--------------------------------------------------
Gary M. Hathaway, Director
PPMAL - Protein/Peptide Micro Analytical Laboratory
California Institute of Technology
139-74, Division of Biology
Pasadena, CA 91125
http://www.cco.caltech.edu/~ppmal
email Gary: hathaway@cco.caltech.edu
email facility: ppmal@cco.caltech.edu
phone: lab (626) 395-6388 or office (626) 395-2769
FAX (626) 449-3414
-------------------------------------------------