Tim,
You just want antibodies, right? So don't knock yourself out doing
well-characterized chemistry. (Lab animals have no appreciation for sexy
chemistry.) Just take any old Tyr-P containing peptide or two and couple a
bunch of it onto your favorite carrier protein using your favorite coupling
technology. Personally I'd recommend a lysine rich Tyr-P peptide, if
available, and simple roar-and-pour glutaraldehyde coupling. People usually
like Megathuria hemocyanin as a carrier, but Limulus hemocyanin is just as
good and often much cheaper.
If you need an immunological reagent with more specificity than that, you're
going to have to do affinity purification, anyway. Appropriate recipes for
conjugation and affinity purification may be found conveniently in the
references below. I know they're kinda old, but the state of this
technology really hasn't progressed much since 1994.
J M Carter (1996) "Conjugation of peptides to carrier proteins via
glutaraldehyde," in The Protein Protocols Handbook, Ed J M Walker, Humana
Press, Totowa, New Jersey, Ch 117 (pp 679-687).
J M Carter (1996) "Conjugation of peptides to carrier proteins via
m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS)," ibid., Ch 118 (pp
689-692).
J M Carter (1996) "Conjugation of peptides to carrier proteins via
carbodiimide," ibid. Ch 119 (pp 693-694).
J M Carter (1996) "Production and characterization of antibodies against
peptides," ibid. Ch 123 (pp 711-716).
J M Carter (1994) "Techniques for conjugation of synthetic peptides to
carrier molecules," in Methods in Molecular Biology, Vol 36 Peptide Analysis
Protocols, ed. B M Dunn and M W Pennington, Humana Press, Totowa, New
Jersey, Ch 10 (pp 155-191).
-- . . . . . Dr. John Mark Carter MCarter@AxCellBio.com . AxCell Biosciences Corporation 826 Newtown-Yardley Road, Suite 100 Newtown, PA 18940-1721 office 267.757.1223 lab 267.757.1230 FAX 267.757.1301 www.AxCellBio.com . . Axcell Biosciences is a wholly owned subsidiary of the Cytogen Corporation. . . . . . "Ohne Analyse, keine Synthese." [Without analysis, no synthesis.] -Friedrich Engels, Anti-D¸hring, 1878-----Original Message----- From: Tetaz, Tim {PRBM~Basel} [mailto:TIM.TETAZ@roche.com] Sent: Tuesday, September 26, 2000 11:11 AM To: Recipients of ABRF List Subject: Phosphotyrosine coupling
Does anybody have a cut and dried method for coupling phosphotyrosine to a protein carrier to use for the production of antibodies? I imagine the best approach would be to chemically block the amino group of the phosphotyrosine, activate the carboxyl group with carbodiimide, couple this entity to the protein and then deprotect the amino group. The removal of the amino group would have to be performed under conditions which didn't denature the protein. Thanks in advance if anyone can help.
Dr Tim Tetaz C/- Hoffman-La Roche Inc. PRBM, Bau 68/327 CH-4070 Basel Switzerland.
Phone: (41) 61 687 1877 Fax: (41) 61 688 2438 Email: tim.tetaz@roche.com
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