Nori Human Osteopontin ELISA Kit Summary

Alternative names for osteopontin: OPN, bone sialoprotein I, BSP-1, BNSP, early T-lymphocyte activation, ETA-1, secreted phosphoprotein 1, SPP1, 2ar and Rickettsia resistance, Ric, BNSP

Standard Curve

Kit Components
1. Pre-coated 96-well Microplate
2. Biotinylated Detection Antibody
3. Streptavidin-HRP Conjugate
4. Lyophilized Standards
5. TMB One-Step Substrate
6. Stop Solution
7. 20 x PBS
8. Assay Buffer

Other Materials Required but not Provided:
1. Microplate Reader capable of measuring absorption at 450 nm
2. Log-log graph paper or computer and software for ELISA data analysis
3. Precision pipettes (1-1000 µl)
4. Multi-channel pipettes (300 µl)
5. Distilled or deionized water

Protocol Outline
1. Prepare all reagents, samples and standards as instructed in the datasheet.
2. Add 100 µl of Standard or samples to each well and incubate 1 h at RT.
3. Add 100 µl of Working Detection Antibody to each well and incubate 1 h at RT.
4. Add 100 µl of Working Streptavidin-HRP to each well and incubate 20 min at RT.
5. Add 100 µl of Substrate to each well and incubate 5-30 min at RT.
6. Add 50 µl of Stop Solution to each well and read at 450 nm immediately.

Background: 

Osteopontin (OPN), also known as bone sialoprotein I (BSP-1 or BNSP), early T-lymphocyte activation (ETA-1), secreted phosphoprotein 1 (SPP1), 2ar and Rickettsia resistance (Ric),^[1] is a protein that is encoded by the SPP1 gene (secreted phosphoprotein 1). The murine ortholog is Spp1. Osteopontin is a SIBLING (glycoprotein) that was first identified in 1986 in osteoblasts. It is expressed in bone, but also in other tissues such as monocytes and macrophages.^[2] Like its name, osteopontin functions as a linking protein and is involved in various cellular processes such as cell activation and apoptosis and various diseases such as cancer, heart disease and inflammatory diseases. Osteopontin is an extracellular structural protein and therefore an organic component of bone. Synonyms for this protein include sialoprotein I and 44K BPP (bone phosphoprotein). Full-length OPN (OPN-FL) can be modified by thrombin cleavage, which exposes a cryptic sequence, SVVYGLR on the cleaved form of the protein known as OPN-R (Fig. 1). This thrombin-cleaved OPN (OPN-R) exposes an epitope for integrin receptors of α4β1, α9β1, and α9β4.^[3] OPN-R can be further cleaved by Carboxypeptidase B (CPB) by removal of C-terminal arginine and become OPN-L. It appears an intracellular variant of OPN (iOPN) is involved in a number of cellular processes including migration, fusion and motility.^[4] Various human cancers, including breast cancer, have been observed to express splice variants of OPN.^[5] The cancer-specific splice variants are osteopontin-a, osteopontin-b, and osteopontin-c. Exon 5 is lacking from osteopontin-b, whereas osteopontin-c lacks exon 4.^[5] Osteopontin-c has been suggested to facilitate the anchorage-independent phenotype of some human breast cancer cells due to its inability to associate with the extracellular matrix.^[5]

References

1. Rangaswami H, et al. (2006). Trends Cell Biol. 16 (2): 79–87.
2. Sodek J, et al. (2006). Dent. Res. 85 (5): 404–15.
3. Laffón A, et al. (1991). Clin. Invest. 88 (2): 546–52.
4. Zohar R, et al. (2000). J Cell Physiol 184 (1): 118–130.
5. He B, et al. (2006). Oncogene 25 (1): 2192–2202.