Article ID | Journal | Published Year | Pages | File Type |
---|---|---|---|---|
9126919 | Gene | 2005 | 10 Pages |
Abstract
Complementary DNA (cDNA) clones for human KIAA genes have been isolated as long cDNAs (>Â 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.
Keywords
GSTPBSAPC2FITCDAPImRNATETkDaORFGFP4′,6-diamidino-2-phenylindole, dihydrochloridecDNADNA complementary to RNAmessenger RNASmall interfering RNAsiRNABasic Local Alignment Search ToolBlastExpression vectorTetracyclineELISAEnzyme-linked immunosorbent assayBase pair(s)Immunofluorescence stainingcytomegalovirusCMVfluorescein isothiocyanateopen reading framePhosphate-buffered salinepolymerase chain reactionPCRMolecular weightWestern blottinggreen fluorescent proteinkilobase(s)kilodaltonsglutathione-S-transferase
Related Topics
Life Sciences
Biochemistry, Genetics and Molecular Biology
Genetics
Authors
Akiyuki Ozaki, Takahiro Nagase, Ayako Watanabe, Daisuke Nakajima, Kiyo Shimada, Mihoko Nagano, Osamu Ohara, Hisashi Koga, Susumu Inamoto,