Utilization of mammalian cells for efficient and reliable evaluation of specificity of antibodies to unravel the cellular function of mKIAA proteins.
Complementary DNA (cDNA) clones for human KIAA genes have been isolated as long cDNAs (>4 kb) with unknown functions. To facilitate the functional analysis of these human clones, we have isolated and determined the structures of their respective mouse homologues (mKIAA genes). Furthermore, we have comprehensively raised antibodies against the translated mKIAA proteins in order to establish a platform for their functional analysis. Since the specificity of these antibodies is critical for subsequent analyses of protein function, here we introduce two assays utilizing mammalian cells to improve their evaluation. First, we have established a semi-high-throughput production of C-terminally FLAG epitope-tagged proteins for Western blotting using specially designed mammalian expression vectors. Secondly, we have utilized immunofluorescence staining of mouse cells to analyze the subcellular localization of endogenous mKIAA proteins. Importantly, these methods allow us to detect potential posttranslational modification of the mKIAA/KIAA proteins and to predict their biological function based on their subcellular localization.
Ozaki
Akiyuki
A
Department of Biotechnology, Institute of Research and Innovation, 1201 Takada, Kashiwa, Chiba 277-0861, Japan.
Nagase
Takahiro
T
Watanabe
Ayako
A
Nakajima
Daisuke
D
Shimada
Kiyo
K
Nagano
Mihoko
M
Ohara
Osamu
O
Koga
Hisashi
H
Inamoto
Susumu
S
D000818:Animals; D000918:Antibody Specificity / Q000276:immunology*; D015153:Blotting, Western; D002460:Cell Line; D018076:DNA, Complementary / Q000235:genetics; D005822:Genetic Vectors; D006801:Humans; D007074:Immunoglobulin G / Q000276:immunology*; D007668:Kidney / Q000196:embryology; D051379:Mice; D009363:Neoplasm Proteins / Q000502:physiology*; D011817:Rabbits; D011994:Recombinant Proteins; D014162:Transfection
D018076:DNA, Complementary; D007074:Immunoglobulin G; D009363:Neoplasm Proteins; D011994:Recombinant Proteins