The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
1/100 - 1/300.
1/1000. at this dilution, the antibody will strongly detect approximately 250 ng of OPN protein on a blot.
1/100 - 1/500. PubMed: 16128620
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.
ICC/IF image of ab8448 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 (ab8448, 1/1000 dilution) 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.
Immunohistochemistry (Frozen sections) - Anti-Osteopontin antibody (ab8448)This image is a courtesy of Anonymous Abreview
ab8448 staining Osteopontin in mouse developing skeleton tissue section by Immunohistochemistry (Frozen sections). Tissue samples were fixed with paraformaldehyde before permeabilization with 0.1% Triton and blocking with 20% serum was performed for 1 hour at RT. The sample was incubated with primary antibody (1/200) in 20%FBS/PBS for 16 hours at 40C. An Alexa Fluor®488-conjugated donkey polyclonal to rabbit IgG was used as secondary antibody at 1/200 dilution. In the image: Red Rhodamine Phalloidin (muscle), Blue DAPI (nuclei), Green Osteopontin.
Breast tumour section. Osteopontin is a normal component of elastic fibers in the breast (shown heavily stained in this section). There is also weak staining of the extracellular matrix. Osteopontin is not believed to be expressed inside breast tumour cells, and there is no staining in the intracellular region of the breast cells in this section.
Osteopontin antibody (ab8448) used at 1:100-1:300. No antigen retrieval is required.
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Osteopontin antibody (ab8448)Image is courtesy of an Abreview submitted by Helder Fonseca
ab8448 staining Osteopontin in Mouse femur tissue sections by Immunohistochemistry (IHC-P - paraformaldehyde-fixed, paraffin-embedded sections). Tissue was fixed with paraformaldehyde and blocked with 5% BSA for 1 hour at 35°C; antigen retrieval was by heat mediation in a citrate buffer. Samples were incubated with primary antibody (1/50) for 14 hours at 4°C. An Alkaline Phosphatase-conjugated Goat anti-rabbit IgG F(ab')2 polyclonal (1/100) was used as the secondary antibody.
Other - Anti-Osteopontin antibody (ab8448)This image is courtesy of Agnihotri et al, JBC 2001
OPN is cleaved by MMP to yield 2 fragments, which migrate at 40kD(N terminal) and 32kD (C terminal). The C terminal fragment can undergo further cleavage by both of these MMPs (see Agnihotri et al, JBC 2001 for further details). The epitope recognised by ab8448 is shown in violet. This antibody detects the full length OPN and the 32kD fragment. It does not recognise the 40kD fragment.