[Preparation and identification of the polyclonal antibody against human VSTM1]

To prepare and characterize the polyclonal antibody against human VSTM1.

VSTM1 has two main isoforms, VSTM1-v1, a type I transmembrane protein, and VSTM1-v2, a classical secretory protein, lacking only the transmembrane domain compared with VSTM1-v1. Two recombinant prokaryotic proteins of VSTM1-v2, Trx-His-S-VSTM1-v2 and GST-VSTM1-v2, were constructed, expressed, purified, and then used for immunization of New Zealand rabbits to prepare anti-VSTM1 antibody and coupling with CNBr-activated Sepharose 4B to purify the antibody by immunoaffinity chromatography, respectively. ELISA was performed to detect the titers of the antiserums. After purification, the antibody was identified by Western blotting and immunofluorescence cytochemistry.

The titers of the antiserums from the two immunized rabbits were both 1:10(6);. Western blotting confirmed that the purified antibody could recognize both the overexpressed and endogenous VSTM1 specifically. Immunofluorescence cytochemistry verified that the antibody could also recognize the overexpressed VSTM1-v1 on the surface of HEK293T cells.

The rabbit antibody against human VSTM1 of a high titer has been obtained, which can be used for recognizing endogenous VSTM1 in immunofluorescence cytochemistry.