Preparation of Polyclonal Antibodies Against the Small G Protein ARL3 and Identification of Its Mutants
To investigate the function of ARL3 in ciliogenesis and ciliary signal transduction,the author generate a rabbit polyclonal antibody against Chlamydomonas reinhardtii ARL3 and to identify arl3 mutants.The target gene,arl3,was obtained through gene cloning,and a prokaryotic expression vector,pET-28a(+)-arl3,was constructed for expression in Escherichia coli BL21(DE3)to produce the fusion protein 6×His-ARL3.The purified target protein was used to immunize rabbits,resulting in the production of antiserum.The antiserum was subsequently purified using protein A and antigen-antibody,and Western blot was employed to detect the presence of the Chlamydomonas reinhardtii ARL3 antigen.Results indicated that the antiserum titer reached 1∶102 400,with the purified ARL3 antibody detecting a single specific band at a dilution of 1∶400.This confirms the successful preparation of a highly specific and sensitive rabbit polyclonal antibody against Chlamydomonas reinhardtii ARL3,as well as the identification of an arl3 mutant.These findings provide a foundational basis for further investigation into the function of ARL3 protein within cilia.
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