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KMID : 1204320080240010105
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Laboratory Animal Research
2008 Volume.24 No. 1 p.105 ~ p.109
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Production and Characterization of Anti-ARHGEF 9 Antibody
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Shin Eun-Young
Kim Chan-Hyung
Kim Yang-Mi
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Abstract
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ARHGEF9 (hPEM2: human homologue of Ascidian protein Posterior End Mark-2, collybistin) is known as a guanine nucleotide exchange factor (GEF) specific for Cdc42, a Rho family GTPase. ARHGEF9 like other GEFs consists of an N-terminal Src homology 3 (SH3), tandem Dbl homology (DH) and pleckstrin homology (PH) domains and C-terminal proline-rich (PR) sequence. But the biological roles of ARHGEF9 have been not clear to date. Therefore, we planed this study to produce the antibody against ARHGEF9 as a tool to investigate the biological functions for the unknown GEF, ARHGEF9. To obtain the antigen, SH3 domain for ARHGEF9 was cloned into a bacterial expression vector (pGEX-4T) and purified by glutathione-affinity chromatography. Purified antigen (0.5 ㎎) was immunized on a rabbit. The production of antibody was determined by Enzyme-Linked Immunosorbent Assay (ELISA) with specific antigen and serum from rabbit. To characterize the antibody, we carried out the western blot analysis in various tissues from rats and protein lysates from some stomach and colon cancer cell lines. We obtained the polyclonal antibody against SH3 domain for ARHGEF9. This antibody specifically recognized the ARHGEF9. We found the expression of ARHGEF9 on brain, kidney, liver, uterus and testis from rats through western blot analysis using anti-ARHGEF9 antibody. And then, ARHGEF9 have also expressed in 6 stomach cancer cell lines and 4 colon cancer cell lines. We produced a polyclonal antibody against ARHGEF9 which is used for the western blot analysis. This antibody might be effective tool for studying biological roles of ARHGEF9.
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KEYWORD
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Guanine nucleotide exchange factor(GEF), ARHGEF9, GTPase
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