title: Expression and purification of human ARP1 protein and rapid preparation of polyclonal antibody.
abstract: Angiopoietin-related protein 1 (ARP1) is one of the antiangiogenic factors and plays an important role in endothelial cell proliferation, migration, and blood vessel network formation. Here a rapid method to prepare ARP1 polyclonal antibody in 1 month was developed. The gene of fibrinogen homology domain (FD) for ARP1 was cloned and the protein was expressed in a soluble form of MBP-FD fused protein. The MBP-FD protein was purified using amylose affinity chromatography of maltose-binding protein. Polyclonal antibodies against MBP-FD were obtained through immunization in BALB/c mice. The titer was determined by indirect enzyme-linked immunosorbent assay (ELISA), and the antibody specificity was assessed by Western blot. The full-length ARP1 protein in stable form expressed in transfected human large lung cancer cell lines NCI-H460 was detected by immunocytochemistry (ICC) analysis using ARP1 polyclonal antibodies. The result shows that the antibody possesses good specificity and sensitivity. This work provides a substantial base for the further studies of ARP1 function and associated mechanisms.   Supplemental materials are available for this article. Go to the publisher's online edition of Preparative Biochemistry and Biotechnology to view the supplemental file.
authors: Department of Biochemistry and Molecular Biology, Second Military Medical University, Shanghai, PR China.
authors: Mingjuan
authors: M
authors: Sun
authors: Rongjiang
authors: R
authors: Zou
authors: Xiaoyi
authors: X
authors: Dong
authors: Ying
authors: Y
authors: Zong
authors: Yun
authors: Y
authors: Gao
authors: Lianghua
authors: L
authors: Wang
authors: Binghua
authors: B
authors: Jiao
mesh_terms: D000091304:Angiopoietin-Like Protein 1; D000075802:Angiopoietin-like Proteins; D042682:Angiopoietins / Q000235:genetics* / Q000276:immunology* / Q000302:isolation & purification*; D000818:Animals; D000906:Antibodies / Q000276:immunology / Q000302:isolation & purification*; D000918:Antibody Specificity; D045744:Cell Line, Tumor; D002846:Chromatography, Affinity; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000235:genetics; D005982:Glutathione Transferase / Q000235:genetics; D006801:Humans; D008175:Lung Neoplasms / Q000235:genetics; D058619:Maltose-Binding Proteins / Q000235:genetics / Q000378:metabolism; D051379:Mice; D008807:Mice, Inbred BALB C; D015202:Protein Engineering / Q000379:methods*; D011994:Recombinant Proteins / Q000235:genetics / Q000276:immunology
chemical_list: C117578:ANGPTL1 protein, human; D000091304:Angiopoietin-Like Protein 1; D000075802:Angiopoietin-like Proteins; D042682:Angiopoietins; D000906:Antibodies; D058619:Maltose-Binding Proteins; D011994:Recombinant Proteins; D005982:Glutathione Transferase