A comprehensive approach for establishment of the platform to analyze functions of KIAA proteins: generation and evaluation of anti-mKIAA antibodies.
Since December 2001 we have been conducting a project to isolate and determine entire sequences of mouse KIAA cDNA clones which encode polypeptides corresponding to human KIAA proteins. The ultimate goal of this project is the elucidation of the functions of KIAA proteins. A critical step in this project is the generation of antibodies based on the cDNA sequence information. Although antibodies are the most optimal tools for biological analysis, the production and isolation of multiple recombinant proteins for an antigen is a rate-limiting step in antibody production. To address this problem, we established a system utilizing the in vitro recombination-assisted method and shotgun clones that were generated during the sequencing of mouse KIAA cDNAs (DNA Res. 2003, 10, 129-136). The authenticity of the expressed proteins was confirmed by matrix-assisted laser desorption/ionization-time of flight mass spectrometry. Another critical step for antibody production is the evaluation of the antibodies. Thus, we also made efforts to develop a systematic approach for evaluation of the titer and the specificity of the antibodies. Using these systems, we have produced and evaluated more than 500 antibodies raised against mouse KIAA proteins to date. We are currently generating antibody arrays for analysis of protein expression profiles. We will verify protein-protein interactions using immunoprecipitation and tandem mass spectrometry analysis.
Koga
Hisashi
H
Kazusa DNA Research Institute, Kisarazu, Chiba, Japan. hkoga@kazusa.or.jp
Shimada
Kiyo
K
Hara
Yasuhiro
Y
Nagano
Mihoko
M
Kohga
Hiroshi
H
Yokoyama
Ryo
R
Kimura
Yayoi
Y
Yuasa
Sigeaki
S
Magae
Junji
J
Inamoto
Susumu
S
Okazaki
Noriko
N
Ohara
Osamu
O
D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies / Q000276:immunology* / Q000378:metabolism*; D000918:Antibody Specificity; D000941:Antigens / Q000276:immunology; D015153:Blotting, Western; D002999:Clone Cells; D018076:DNA, Complementary / Q000235:genetics*; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D005620:Freund's Adjuvant / Q000276:immunology; D007074:Immunoglobulin G / Q000302:isolation & purification; D007279:Injections, Subcutaneous; D013058:Mass Spectrometry; D051379:Mice; D008813:Mice, Inbred ICR; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D011506:Proteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology* / Q000378:metabolism*; D011817:Rabbits; D011994:Recombinant Proteins / Q000378:metabolism; D015203:Reproducibility of Results; D017386:Sequence Homology, Amino Acid; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014357:Trypsin
D000906:Antibodies; D000941:Antigens; D018076:DNA, Complementary; D007074:Immunoglobulin G; D010446:Peptide Fragments; D011506:Proteins; D011994:Recombinant Proteins; D005620:Freund's Adjuvant; D014357:Trypsin