The preparation of anti-CLC-7 polyclonal antibody

Objective To prepare the polyclonal antibody against human chloride channel 7(CLC-7). Methods EGFP-CLC-7 fusion protein was expressed in E. coli by transforming the bacteria DH-5α with the construct pEGFP-CLC-7.The expression was induced by IPTG for 24 h. Bacterial proteins were extracted and subjected to SDS-PAGE electrophoresis.The protein of interest was identified on the gel of SDS-PAGE according to the molecular weight.The gel containing the separated protein of the interest was collected, minced, and mixed with immune adjuvant BCG.New Zealand white rabbits were immunized with 1g protein extract mixed with BCG for five times in 6 weeks.Sera of the immunized rabbits were collected.The specificity and the titer of the antibody against CLC-7 were determined by Western blot. Results The prepared antibody against human CLC-7 showed the same pattern in the reaction with CLC-7 expressed in both E. coli and HeLa cells as the commercially available CLC-7 polyclonal antibody (obtained from Abcam). The titer of the prepared polyclonal antibody against CLC-7 reached to 1∶4 000. Conclusion Polyclonal antibody against human CLC-7 was obtained by immunizing rabbit with CLC-7 expressed in E. coli.This antibody, with titration up to 1∶4000, showed high specificity compared to the commercially available CLC-7 antibody. Key words: Chloride channel 7; Antibody