Production of Affinity-Purified Polyclonal Antibody Against Recombinant TIS21 Protein

Abstract

For the study of TIS21 protein, a primary response gene product, we produced affinity-purified antisera against recombinant TIS21 protein. At first, hexahistidine-tagged recombinant TIS21 protein was produced by using bacterial expression system and was purified to near homogeneity using Ni2+ -affinity column in the presence of urea. PBS-soluble and -insoluble TIS21 protein fractions were immunized into rabbits after dialysis. High-titer antiserum was obtained from one of the rabbits immunized with PBS-soluble fraction. Meanwhile, partially purified recombinant TIS21 protein was further purified by preparative SDS-PAGE and it was conjugated with the sepharose resin. The high-titer serum was applied to this affinity column and affinity -purified antiserum was obtained. This affinity-purified antiserum recognized TIS21 proteins expressed in bacteria as well as in mammalian cell when it was used in western blot. In addition, this affinity-purified antiserum recognized a constitutively expressed 40 kDa protein from NIH3T3, B9, C3HIOT1/2 and 293 cell lines. Detection of this protein with the affinity-purified antiserum can be inhibited by recombinant TIS21 proteins prepared by our laboratory and other laboratory. Therefore this 40kDa protein can be an immunologically cross-reactive protein with TIS21 protein.