Generation of a polyclonal antibody that simultaneously detects multiple Ser/Thr protein kinases

Journal of Biochemical and Biophysical Methods
Isamu KameshitaAtsuhiko Ishida

Abstract

In order to obtain a polyclonal antibody that recognizes various protein kinases, a peptide corresponding to an amino acid sequence of a highly conserved subdomain (subdomain VIB) of the protein kinase family was synthesized and used for immunization. When the synthetic peptide, CVVHRDLKPENLLLAS, was coupled to keyhole limpet hemocyanin (KLH) and used to immunize rabbits, polyclonal antibodies that detected multiple protein kinases on a Western blot were generated. One of the antibodies obtained, KI98, detected a variety of purified Ser/Thr protein kinases, such as calmodulin-dependent protein kinase II (CaM-kinase II), calmodulin-dependent protein kinase IV (CaM-kinase IV), cAMP-dependent protein kinase, protein kinase C, and Erk2. The antibody detected as low as 0.2 ng of protein kinases blotted onto a nitrocellulose membrane by dot-immunobinding assay. When a rat brain extract was analyzed with this antibody, various protein kinases were simultaneously detected. The present anti-peptide antibody with a broad spectrum of cross-reactivity to multiple protein kinases may be a powerful tool for comprehensive analysis focused on protein kinases.

References

Jan 1, 1976·Analytical Biochemistry·A Bensadoun, D Weinstein
Feb 3, 1995·The Journal of Biological Chemistry·A Ishida, H Fujisawa
Jun 1, 1994·Journal of Biochemistry·A IshidaH Fujisawa

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Citations

Sep 28, 2007·Biochemical and Biophysical Research Communications·Noriyuki SueyoshiAtsuhiko Ishida
Jan 5, 2007·Journal of Pharmacological Sciences·Atsuhiko IshidaYasushi Shigeri
Apr 21, 2018·Bioscience, Biotechnology, and Biochemistry·Masashi YamashitaYasushi Shigeri
Dec 16, 2006·Archives of Biochemistry and Biophysics·Takaki NimuraIsamu Kameshita
May 15, 2007·Archives of Biochemistry and Biophysics·Sachiko ShimomuraIsamu Kameshita

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