[Generation and preliminary application of rabbit anti-human polyclonal antibodies against NH2-terminal peptides of CXCR3-B].
ABSTRACT To generate and identify the rabbit polyclonal antibodies against NH(2)-terminal peptides of CXCR3-B.
Three peptides (aa. 1 to 19, aa. 17 to 35, and aa. 33 to 51) of human CXCR3-B NH(2)-terminus were synthesized by using standard Fmoc. The synthesized peptides were purified by reversed phase high-performance liquid chromatography (RP-HPLC), and cross-linked with keyhole limpet hemocyanin (KLH) by sodium metaperiodate. Rabbits were immunized with conjugated peptides for 3 times (400 microg/rabbit). The polyclonal antibodies were purified by Protein G from the collected antiserum.
NH(2)-terminal peptides of human CXCR3-B with the purity of 98%, 88.54%, and 80%, respectively, were prepared. The titers of purified polyclonal antibodies were 1:32,000 (0.1 mg/L), 1:4,000 (3 mg/L), and 1:1,000 (10 mg/L), respectively. Western blot results showed that the antibodies could recognize the protein with molecular weight of 50,000 in the total lysates of human fetal heart, whereas the antibodies against the peptides of No.1-19 amino acids could also recognize an additional protein (40,000). The antigens recognized by the antibodies were localized in the vascular endothelial cells of human fetal heart tissues.
The polyclonal antibodies against NH(2)-terminal peptides generated and identified in the present work are specific to CXCR3-B protein and therefore can be useful tools for the functional study of human CXCR3-B.