Anti his c term antibody anti his tag mab

The recombinant (Bβ1–66)₂ fragment was produced in E. coli and purified as previously described^{9 (link)}. This fragment was treated with thrombin to produce fibrin-related (β15–66)₂ fragment using the previously described procedure^{21 (link)}. Human recombinant receptor-associated protein (RAP) was expressed in E. coli and purified as previously described^{22 (link)}. Mouse anti-hVLDLR monoclonal antibodies 1H5, 5F3, and 1H10^{23 (link)} were kindly provided by Dr. D. Strickland (University of Maryland Baltimore). Anti-His(C-term) antibody (anti-His tag mAb) conjugated with HRP was from Invitrogen (Carlsbad, CA). Goat secondary anti-mouse polyclonal antibodies conjugated with HRP and HRP substrate SureBlue TMB were from KPL (Gaithersburg, MD). Cyanogen bromide-activated Sepharose 4B and Thrombin CleanCleave kit were obtained from Sigma (St. Louis, MO).

NDSK-II fragment was prepared by digestion of human fibrinogen (Enzyme Research Laboratories) with CNBr followed by cleavage of its fibrinopeptides with thrombin-agarose as described earlier (8 (link), 20 (link)). Human receptor-associated protein (RAP) was expressed in E. coli and purified as described (21 (link)). Anti-human VLDLR monoclonal antibodies (mAb) 1H5, 1H10, and 5F3 (18 (link)) were purified from hybridoma supernatants by affinity chromatography on Protein A-Sepharose (Sigma-Aldrich). All three monoclonal antibodies cross-reacted with mouse VLDLR as revealed by immunostaining of mouse heart tissue sections. Anti-VLDLR mAb E8 and 6A6 and anti-β-tubulin mAb G-8 were obtained from Santa Cruz Biotechnology. Purified mouse IgG1, κ isotype control antibody, was from Biolegend. Goat secondary anti-mouse antibodies conjugated with HRP and HRP substrate SureBlue TMB were from KPL. The anti-His(C-term) antibody (anti-His tag mAb) conjugated with HRP was from Invitrogen. Calcein AM fluorescent dye, phorbol 12-myristate 13-acetate (PMA), and N-formyl-Met-Leu-Phe (fMLP) were obtained from BD Biosciences, Promega, and Sigma-Aldrich, respectively.