This antibody gave a positive signal in the following lysates:
Rat Skeletal Muscle Tissue
Rat Brain Tissue
HL60 Whole Cell
Mouse Brain Tissue
Rat Liver Tissue (data not shown)
Mouse Liver Tissue (data not shown)
Human Liver Tissue (data not shown)
Shipped at 4°C. Store at +4°C short term (1-2 weeks). Upon delivery aliquot. Store at -20°C or -80°C. Avoid freeze / thaw cycle.
The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
Use a concentration of 5 µg/ml.
Use a concentration of 5 µg/ml. Perform heat mediated antigen retrieval before commencing with IHC staining protocol.
Use a concentration of 1 µg/ml. Detects a band of approximately 32 kDa (predicted molecular weight: 32 kDa).
Binds tightly to hydroxyapatite. Appears to form an integral part of the mineralized matrix. Probably important to cell-matrix interaction. Acts as a cytokine involved in enhancing production of interferon-gamma and interleukin-12 and reducing production of interleukin-10 and is essential in the pathway that leads to type I immunity.
Bone. Found in plasma.
Belongs to the osteopontin family.
Extensively phosphorylated on clustered serine residues. N- and O-glycosylated. Phosphorylation sites are present in the extracelllular medium.
IHC image of Osteopontin staining in human breast carcinoma formalin fixed paraffin embedded tissue section, performed on a Leica BondTM system using the standard protocol F. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20 mins. The section was then incubated with ab63856, 5µg/ml, for 15 mins at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX.
ICC/IF image of ab63856 stained MCF7 cells. The cells were 4% PFA fixed (10 min) and then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1h to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab63856, 5µg/ml) overnight at +4°C. The secondary antibody (green) was Alexa Fluor® 488 goat anti-rabbit IgG (H+L) used at a 1/1000 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (red) at a 1/200 dilution for 1h. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43µM.
Western blot - Anti-Osteopontin antibody (ab63856)
All lanes : Anti-Osteopontin antibody (ab63856) at 1 µg/ml
Lane 1 : Skeletal Muscle (Rat) Tissue Lysate - normal tissue (ab29376) Lane 2 : Brain (Rat) Tissue Lysate - normal tissue Lane 3 : HL60 (Human promyelocytic leukemia cell line) Whole Cell Lysate (Human) Whole Cell Lysate Lane 4 : Brain (Mouse) Tissue Lysate
Lysates/proteins at 10 µg per lane.
Secondary All lanes : Goat polyclonal to Rabbit IgG - H&L - Pre-Adsorbed (HRP) at 1/3000 dilution
Performed under reducing conditions.
Predicted band size: 32 kDa Observed band size: 32 kDa Additional bands at: 100 kDa, 42 kDa, 56 kDa, 88 kDa. We are unsure as to the identity of these extra bands.
Yan Y et al. Effect of Thyrotropin on Osteopontin, Integrin avß3, and VCAM-1 in the Endothelium via Activation of Akt. Int J Mol Sci17:N/A (2016).
Read more (PubMed: 27657042) »
Kim MO et al. Electromagnetic fields and nanomagnetic particles increase the osteogenic differentiation of human bone marrow-derived mesenchymal stem cells. Int J Mol Med35:153-60 (2015).
Read more (PubMed: 25352086) »