Generation of a polyclonal antibody that simultaneously detects multiple Ser/Thr protein kinases.
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.
Kameshita
Isamu
I
Faculty of Agriculture, Department of Life Sciences, Kagawa University, Miki-cho, Kagawa 761-0795, Japan. kamesita@ag.kagawa-u.ac.jp
Kinoshita
Shun
S
Shigeri
Yasushi
Y
Tatsu
Yoshiro
Y
Yumoto
Noboru
N
Ishida
Atsuhiko
A
D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies / Q000737:chemistry*; D015153:Blotting, Western; D001921:Brain / Q000378:metabolism; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D054734:Calcium-Calmodulin-Dependent Protein Kinase Type 4; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism; D002417:Cattle; D003101:Collodion / Q000737:chemistry; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D006433:Hemocyanins / Q000737:chemistry; D006801:Humans; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism; D008969:Molecular Sequence Data; D009206:Myocardium / Q000378:metabolism; D010455:Peptides / Q000737:chemistry; D020033:Protein Isoforms; D011493:Protein Kinase C / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry* / Q000378:metabolism; D011817:Rabbits; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry; D012680:Sensitivity and Specificity
D000906:Antibodies; D010455:Peptides; D020033:Protein Isoforms; D011994:Recombinant Proteins; D003101:Collodion; D006433:Hemocyanins; D017346:Protein Serine-Threonine Kinases; D017868:Cyclic AMP-Dependent Protein Kinases; D011493:Protein Kinase C; C517683:CAMK4 protein, human; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D054734:Calcium-Calmodulin-Dependent Protein Kinase Type 4; D017871:Calcium-Calmodulin-Dependent Protein Kinases; C517686:Camk4 protein, rat; D019950:Mitogen-Activated Protein Kinase 1; C032808:keyhole-limpet hemocyanin