This mouse IgG2a monoclonal antibody (clone AK1G4) was generated against purified recombinant human Osteopontin (OPN) and is specific for the C-terminal region of human OPN (epitope: aa 13-16).
Osteopontin (OPN, also designated Bone Sialoprotein 1, Urinary Stone Protein, spp-1, eta-1, nephropontin, uropontin) is a phosphoglycoprotein synthesized in a variety of tissues and cells. It was originally identified as a bone matrix protein and subsequently as a cytokine produced by various immune and transformed cell lines. OPN is a biomarker for various types of cancers and inflammatory diseases. Excessive or dysregulated OPN expression has been linked to the pathogenesis of both autoimmune disorders such as multiple sclerosis, systemic lupus erythematosus, rheumatoid arthritis, atherosclerosis and other inflammatory diseases including cardiovascular disease, chronic obstructive pulmonary disease, inflammatory bowel disease, liver disease and asthma.
From the laboratory of David T. Denhardt, PhD, Rutgers University .
Part of The Investigator's Annexe program.
|Antigen:||Osteopontin (OPN) protein|
|Reactivity:||Human and mouse|
|Immunogen:||recombinant histidine-tagged human OPN (prepared in E. coli and purified on a nickel column)|
|Purification Method:||Protein G|
|Buffer:||0.1M Sodium Phosphate, pH 7.4, 0.15M NaCl, 0.05% (w/v) Sodium Azide|
|Tested Applications:||WB, ELISA, IP|
mAb recognition of OPN in Western Blots
A: Western blots showing antibody recognition of murine OPN. Conditioned media (10 ul/lane) from various cell lines or 50 ng of recombinant murine OPN (GST-mOPN) were separated on 12% SDSPAGE gels and transferred to PVDF membranes. The membranes were then cut into strips which were blotted with monoclonal antibodies at 1 ug/ml or polyclonal control (shown above each lane). 275-3-2: ras-transformed murine fibroblast cell line. 275: non-transformed murine fibroblast 3T3 cell line. MC3T3E1: preosteoblast cell line induced to differentiate for 12 days. B: Western blot of human urine detected with anti-OPN mAbs. Urine was collected and dialyzed extensively against 0.1M NaCl before approximately tenfold concentration with Centriprep spin columns. Five microliters of the concentrated, dialyzed urine was assayed via SDSPAGE and Western blotting with mAbs at 1 ug/ml. Polyclonal antibody LF124 (kindly provided by Dr. Larry Fisher, NIH) and polyclonal anti-OPN (recombinant) mouse serum were used as controls.
Adapted from: Wang KX, et al. J Immunol. 2009 Feb 15;182(4):2485-91.
Antibodies 3D9 and 7B4 inhibit cell adhesion to recombinant OPN
Tissue culture treated 96-well plates were coated with 150 uM human recombinant his-tagged OPN, then blocked with 1% BSA. Antibodies were then added at 125 uM and allowed to bind OPN for 2 h. The wells were then washed and 5 x 104 MDA-MB-435 (A) or 275-3-2 (B) cells were added and allowed to adhere for 3 or 3.5 h respectively. Non-adherent cells were removed by washing and adherent cells were quantitated by staining with crystal violet. Data are representative of four independent experiments for the MDA-MB-435 cell line and two independent experiments for the 275-3-2 cell line (n=4). *P<0.001 Students t-test.
Adapted from: Kazanecki CC, et al. J Cell Biochem. 2007 Nov 1;102(4):925-35.
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