Prokaryotic Expression,Purification and Polyclonal Antibody Preparation of Small G Protein ARL1 of Chlamydomonas reinhardtii
In order to study the role of small G protein ARL1 in ciliary formation and ciliary signal transduction,a highly specific polyclonal antibody against ARL1 of Chlamydomonas reinhardtii was prepared.First,6×His labeled prokaryotic expression vector pET-28a(+)-arl1 was constructed,and the fusion protein 6×His-ARL1 was induced in E.coli DH5α/C+,and then purified and immunized New Zealand male white rabbits.After three times of immunization,the serum of auricular artery was taken,and the titer was tested by indirect ELISA.ARL1 antibody with high sensitivity and specificity was ob-tained by affinity purification of Protein A and antigenic antibody.By immunofluorescence and Western blot detection,ARL1 was found to be localized in cytoplasm,basal body and cilia,which has laid a foundation for further study on the func-tion of ARL1 in cilia generation and signal transduction of C.reinhardtii.
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