Osteopontin (SPP1) antibody

Antigen: Osteopontin (SPP1)

Synonyms: OPN, BNSP, BSPI, ETA-1, MGC110940

Clonality: Polyclonal

Host: Rabbit

Reactivity: Human, Pig (Porcine), Dog (Canine)

Application: ELISA, Western Blotting (WB), Immunohistochemistry (Paraffin-embedded Sections) (IHC (p))

Catalog no.: ABIN105163

Additional Information

Alternative name: Osteopontin

Immunogen: This whole rabbit serum was prepared by repeated immunizations with a synthetic peptide C-K-S-K-K-F-R-R-P-D-I-Q-Y-P-D corresponding to human osteopontin proteins conjugated to KLH using maleimide. A residue of cysteine was added to the amino terminal end to facilitate coupling.

Format: Liquid (sterile filtered)

Description: Osteopontin (OPN) is an arginine-glycine-aspartic acid (RGD)-containing glycoprotein that interacts with integrins and CD44 as major receptors. OPN is multifunctional, with activities in cell migration, cell survival, inhibition of calcification, regulation of immune cell function, and control of tumor cell phenotype. The gene encoding OPN is called spp1. Targeting this gene has revealed that while OPN is not necessary for normal embryonic development, fertility, and health under pathogen-free conditions, loss of the protein has significant consequences in several models of injury/disease as diverse as renal injury, viral and bacterial infection, bone remodeling, and tumor growth. The fact that no other proteins seem to share a redundant activity with OPN under these conditions suggests that OPN has a unique functional role during tissue injury and stress. Interestingly, several members of the matrix metalloproteinase (MMP) family are also induced during injury/disease processes in patterns overlapping that of OPN. OPN has recently been shown to be a novel substrate for two MMPs, MMP-3 (stromelysin-1) and MMP-7 (matrilysin). There are three cleavage sites for MMP-3 in human OPN, two of which are also cleaved by MMP-7 (see cleavage diagram). Biological assays demonstrate that the MMP-cleaved OPN has increased activity in promoting both cell adhesion and migration compared with full-length OPN. In addition, inhibitory reagents were used to show that the same receptors that interact with OPN also mediate interaction of MMP-cleaved OPN with tumor cells. It is suggested that active forms of OPN at sites of tissue injury may be regulated by the activity of proteases including MMPs and that the differences in activity of modified OPN may be explained by differences in binding affinity of integrins or distinct downstream signaling events.

Specificity: This antiserum is directed against human osteopontin. The antibody recognizes the full length osteopontin protein (which runs at 66 kD on westerns), as well as the C-terminal fragments of both thrombin and MMP-cleaved OPN. The 32 kD MMP-cleaved C-fragment is recognized, but not the 40 kD N-terminal fragment. Cross reactivity occurs with osteopontin from swine and dog. Reactivity with osteopontin from other sources has not been determined.

Application Details

Application Notes: Recommended Dilutions: This product was assayed by immunoblot against recombinant human osteopontin. The antibody exclusively recognizes C-terminal fragments of both thrombin and MMP-cleaved OPN. A 1:1000 dilution will detect strongly approximately 250 ng of OPN protein on a blot. For IHC (formalin-fixed paraffin-embedded) a 1:100 to 1:300 dilution is recommended. No pretreatment is required. Researchers should determine optimal titers for other applications. Researchers should determine optimal titers for other applications. Note: This antibody is suitable for western blotting, immunohistochemistry (formalin-fixed paraffin-embedded sections) and ELISA.

Concentration: 85 mg/ml (by Refractometry)

Buffer: Stabilizer: None. Preservative: 0.1% (w/v) Sodium Azide. Buffer: 0.02 M Potassium Phosphate, 0.15 M Sodium Chloride, pH 7.2.

Storage: Store vial at -20°C prior to opening. For extended storage, aliquot contents and freeze at -20°C or below. Avoid cycles of freezing and thawing. Centrifuge product if not completely clear after standing at room temperature. This product is stable for several weeks at 4°C as an undiluted liquid. Dilute only prior to immediate use. Expiration date is one (1) year from date of opening.

Research Area: Cancer, Extracellular Matrix, Hormones, Enzymes, Metabolism

Restrictions: For Research Use only

Images

Immunohistochemistry. Rabbit anti-Osteopontin was used at a 1:100-1:300 dilution to detect osteopontin by immunohistochemistry. Osteopontin is a normal component of elastic fibers in the breast (shown heavily stained in this section of human breast tumor cells). There is also weak staining of the extracellular matrix. Osteoponin is not expressed in breast tumor cells, and there is no staining of the breast cells in this section. No antigen retrieval is required.

Immunoblotting. Rabbit anti-Osteopontin was used at a 1:1,000 dilution to detect Osteopontin by Western blot. In lane 2 reactivity is shown against 250 ng of human osteopontin. Lane 3 shows reactivity of MMP-cleaved osteopontin. Lane 1 shows the position of molecular weight markers. Use a 1:10,000 dilution of HRP conjugated Gt-a- Rabbit IgG (ABIN102010) for detection.

Osteopontin cleavage diagram. 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. The epitope recognized by Rabbit-anti-Osteopontin is shown in violet. This antibody detects the full length OPN and the 32kD fragment. It does not recognize the 40kD fragment.

Publications

Publications: Denhardt, Chambers: "Overcoming obstacles to metastasis--defenses against host defenses: osteopontin (OPN) as a shield against attack by cytotoxic host cells." in: Journal of cellular biochemistry, Vol. 56, Issue 1, pp. 48-51, 1995 (PubMed).

Denhardt, Lopez, Rollo et al.: "Osteopontin-induced modifications of cellular functions." in: Annals of the New York Academy of Sciences, Vol. 760, pp. 127-42, 1995 (PubMed).

Weber, Cantor: "The immunology of Eta-1/osteopontin." in: Cytokine & growth factor reviews, Vol. 7, Issue 3, pp. 241-8, 1997 (PubMed).

Uede, Katagiri, Iizuka et al.: "Osteopontin, a coordinator of host defense system: a cytokine or an extracellular adhesive protein?" in: Microbiology and immunology, Vol. 41, Issue 9, pp. 641-8, 1997 (PubMed).

Liaw, Birk, Ballas et al.: "Altered wound healing in mice lacking a functional osteopontin gene (spp1)." in: The Journal of clinical investigation, Vol. 101, Issue 7, pp. 1468-78, 1998 (PubMed).

Rittling, Matsumoto, McKee et al.: "Mice lacking osteopontin show normal development and bone structure but display altered osteoclast formation in vitro." in: Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research, Vol. 13, Issue 7, pp. 1101-11, 1998 (PubMed).

Crawford, Matrisian, Liaw: "Distinct roles of osteopontin in host defense activity and tumor survival during squamous cell carcinoma progression in vivo." in: Cancer research, Vol. 58, Issue 22, pp. 5206-15, 1998 (PubMed).

Yoshitake, Rittling, Denhardt et al.: "Osteopontin-deficient mice are resistant to ovariectomy-induced bone resorption." in: Proceedings of the National Academy of Sciences of the United States of America, Vol. 96, Issue 14, pp. 8156-60, 1999 (PubMed).

Noiri, Dickman, Miller et al.: "Reduced tolerance to acute renal ischemia in mice with a targeted disruption of the osteopontin gene." in: Kidney international, Vol. 56, Issue 1, pp. 74-82, 1999 (PubMed).

Nau, Liaw, Chupp et al.: "Attenuated host resistance against Mycobacterium bovis BCG infection in mice lacking osteopontin." in: Infection and immunity, Vol. 67, Issue 8, pp. 4223-30, 1999 (PubMed).

Ophascharoensuk, Giachelli, Gordon et al.: "Obstructive uropathy in the mouse: role of osteopontin in interstitial fibrosis and apoptosis." in: Kidney international, Vol. 56, Issue 2, pp. 571-80, 1999 (PubMed).

Ashkar, Weber, Panoutsakopoulou et al.: "Eta-1 (osteopontin): an early component of type-1 (cell-mediated) immunity." in: Science (New York, N.Y.), Vol. 287, Issue 5454, pp. 860-4, 2000 (PubMed).

McCawley, Matrisian: "Matrix metalloproteinases: multifunctional contributors to tumor progression." in: Molecular medicine today, Vol. 6, Issue 4, pp. 149-56, 2000 (PubMed).

Agnihotri, Crawford, Haro et al.: "Osteopontin, a novel substrate for matrix metalloproteinase-3 (stromelysin-1) and matrix metalloproteinase-7 (matrilysin)." in: The Journal of biological chemistry, Vol. 276, Issue 30, pp. 28261-7, 2001 (PubMed).

Shin, Ahn, Kim et al.: "Temporal expression of osteopontin and CD44 in rat brains with experimental cryolesions." in: Brain research, Vol. 1041, Issue 1, pp. 95-101, 2005 (PubMed).

Kim, Shin: "Immunohistochemical study of osteopontin in boar testis." in: Journal of veterinary science, Vol. 8, Issue 2, pp. 107-10, 2007 (PubMed).

Iwanaga, Ueno, Ueki et al.: "The expression of osteopontin is increased in vessels with blood-brain barrier impairment." in: Neuropathology and applied neurobiology, Vol. 34, Issue 2, pp. 145-54, 2008 (PubMed).