John,
(I used to know all this stuff....line from Laugh-In)
The higher weight is likely due to a couple of factors.
(1) The first and most significant is lyophilization does not remove bound
water. To get true dry weight, the protein must be heated at 110C over a
dessicant such as phosphorus pentoxide in a vacuum oven until constant
weight is obtained, but short of charring. For highest accuracy, the dried
material should be cooled in a tightly closed dessicator and timed
weighings made (Hunter, M. (1966), J. Phys. Chem. 70, 3285-. The weight is
graphed as a function of time and then extrapolated to zero time.
(2) The second problem is the Donnan equilibrium. To get true protein
weight, you have to deal with isoionic protein. If you dialyze the protein
away from its isoelectric point, you will accumulate ions within the
protein solution to equalize charge and are then dealing with a
protein-counter ion (the so-called isopotential) complex. Then you will
have to measure your U.V. absorbance for this defined protein complex which
will require preparing the protein under the same dialysis conditions every
time.
Best of luck,
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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@caltech.edu
email facility: ppmal@caltech.edu
phone: lab (818) 395-6388 or office (818) 395-2769
FAX (818) 449-3414
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