Preparation and identification of rabbit polyclonal antibody against human FAM21

Objective: To prepare rabbit anti human FAM21 polyclonal antibodies and analyze antibody specificity. Method: Using the expression plasmid encoding the full-length FAM21 gene as a template, the nucleotide sequence of 2431-3006 bases was amplified by PCR and connected to the pGEX-6p-1 prokaryotic expression vector to construct the pGEX6p-1-FAM21 recombinant expression plasmid expressing the amino acid fragment 811-1002 of FAM21. Transform the recombinant plasmid into BL21 (DE3) competent Escherichia coli and induce expression. Purify the protein using GST fusion protein and magnetic beads. The purified GST fusion protein was used as an antigen to immunize New Zealand rabbits, and the collected serum was purified using an agarose column containing GST protein. Western blot and immunofluorescence assays were used to detect the specificity of antibodies in HeLa cells with FAM21 gene silencing. Result: The pGEX-6p-1-FAM21 prokaryotic expression plasmid was successfully constructed and induced for expression in BL21 (DE3) Escherichia coli. The purified GST fusion protein had a molecular weight of approximately 50kDa, and the antibody titer purified after immunization with New Zealand rabbits was greater than 1:128000, with high specificity. Conclusion: The pGEX-6p-1-FAM21 prokaryotic expression plasmid was successfully constructed, and rabbit anti human FAM21 polyclonal antibodies were prepared for Western blot and immunofluorescence detection.