Baf53 Polyclonal Antibody Preparation And Activity Of Man

Antibody is one of the most important reagents commonly used in immunological cell chemistry technology. Actually nowadays highly specific and sensitive antibodies are of vital importance to any molecular biological experiment. Currently commercially available specific antibodies as lab reagents are turning up fast and many labs make use of those commercial antibodies. However, in China now only a few labs are trying to make their own antibodies which are very essential for the time being. Especially in the case that a new protein (as antigen) is to be defined, it is better to have a self-made antibody as experimental reagent. hBAF53 is a newly found subunit of a novel chromatin remodeling complex梙uman SWI/SNF complex.To acquire the polyclonal antibody of hBAF53, we made use of several molecular cloning methods. First we amplified BAF53 cDNA from a plasmid using PCR, and then selected a fragment by restriction digestion and made an expression construct of BAF53 fragment. The sequencing result showed that fragment inserted into the expression construct is correct (without mutation). After that the construct is transformed in to E. coli. And the expression is induced by IPTG. Pure hBAF53 fragments were obtained by collecting and lyzing the bacteria and the protein purification followed. The hBAF53 fragment obtained was used as antigens to immunize a rabbit. In the first immunization, equal volume of Freund' s Complete Adjuvant was mixed with antigen solution to enhance the antigenicity. After 2 times of strengthening immunization, the serum of the rabbit was acquired.The activity of the antiserum was tested by dot enzyme immunization assay (DIBA) with purified antigen. Western blot of Hela nuclear protein extract, which contained natural hBAF53, showed that the antiserum is also a specific antiserum to natural hBAF53. Therefore the highly specific and sensitive antiserum can be applied to various studies.