Novagen kit for sequencing immunoglobulins

Cathy Lawson lawson at BNLCL6.BNL.GOV
Thu Jan 26 17:40:31 EST 1995


Dear Immunology netters:

I am a proteins crystallographer who has run into an immunology/molecular
biology problem. In particular, it is an antibody sequencing problem.  If
you have any suggestions please e-mail me directly since I do not subscribe
to the immunology net.

We have been using the Novagen primer kit to try to obtain the sequences of
the heavy and light chains of an antibody Fab that we have managed to
cocrystallize with our real protein of interest (of course, the protein
would not crystallize on its own, thus our predicament).  We have
successfully obtained the heavy chain sequence, but are having no luck with
the light chain: twice we obtained completely unrelated sequences.  Our
questions are:

(1) has anyone else ever had this result with the Novagen kit?  (How) did
you solve the problem?

(2) Total RNA was used to try to fish out the light chain sequence with the
Novagen cDNA primer--would we be better off purifying mRNA first?  

We would be grateful for any helpful hints--

Cathy

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Catherine L. Lawson                      office:      (516) 282 - 7667
Biology Department                       lab:         (516) 282 - 3370
Brookhaven National Laboratory           fax:         (516) 282 - 3407
Building 463                             alternate address: lawson at bnl.gov
Upton, NY 11973
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