May 26, 1994
Hello folks.
Has any one determined the concentration of IgG isotypes in the serum of
adult BALB/cByJ mice? We have spoken with a few people at Jackson
Laboratories, from whom we purchased these animals, but they have no data
even though they get this query from time to time.
The reason for my curiosity is that I am fractionating IgG on protein-A
sepharose and my yields are very low (10-20%) compared to published levels
for BALB/c (not necessarily the ByJ subline). I have quantified the protein
concentrations of the eluted IgG fractions by absorbance at 280 nm and
confirmed this by Bradford assay using purified rat IgG as a standard. I
have also reapplied the application and wash, washed and eluted but got no
more IgG eluting. The column is quite simple - a 5 ml syringe plugged with
glass wool- and run through the detector of our FPLC. I do use the sample
loop and pumps to run the column but there doesn't seem to be any major
retention of protein in the system as evidenced by the visually quantitative
recovery of pigmented material from the starting serum (presumably some
hemoglobin, etc.). The eluted protein as well as the application and wash
have been concentrated with centricon 30 units and there doesn't seem to be
any loss with them.
I have also run a few antibody specific ELISAs with the starting serum and
fractionated isotypes for relative concentration and for the degree of
isotype separation. These assays indicate that I have recovered about 75% of
the specific activity (adding up the contributions of the different isotype
fractions) but there is this nagging question of where has all the protein
gone or where is it hiding??
If any one has any information, ideas or suggestions please let me know by
either e-mail or this BB, etc.
Thanks in advance.
T.
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THOMAS M. DALY / / / / DEPT. OF MICRO. & IMMUNO. MS 405
Voice (215) 762-1903 /___/ / / HAHNEMANN UNIVERSITY
Fax (215) 762-8075 / / / / BROAD & VINE STS.,
DALYT at hal.hahnemann.edu / / /___/ PHILA., PA 19102
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