Description
Introduction: Bone sialoprotein (BSP) is a component of mineralized tissues such as bone, dentin, cementum and calcified cartilage. BSP is a significant component of the bone extracellular matrix and has been suggested to constitute approximately 8% of all non-collagenous proteins found in bone and cementum. BSP was originally isolated from bovine cortical bone as a 23-kDa glycopeptide with high sialic acid content. Native BSP has an apparent molecular weight of 60-80 kDa based on SDS-PAGE, which is a considerable deviation from the predicted weight (based on cDNA sequence) of approximately 33 kDa. The mammalian BSP cDNAs encode for proteins averaging 327 amino acids, which includes the 16-residue preprotein secretory signal peptide. Among the mammalian cDNAs currently characterized, there is an approximate 45% conservation of sequence identity and a further 10-23% conservative substitution. The protein is highly acidic (pKa of ~ 3.9) and contains a large amount of Glu residues, constituting ~22% of the total amino acid.
Principle of the Assay: The microtiter plate provided in this kit has been pre-coated with an antibody specific to BSP. Standards or samples are then added to the appropriate microtiter plate wells with a biotin-conjugated antibody preparation specific for BSP and Avidin conjugated to Horseradish Peroxidase (HRP) is added to each microplate well and incubated. Then a TMB (3,3',5,5' tetramethyl-benzidine) substrate solution is added to each well. Only those wells that contain BSP, biotin-conjugated antibody and enzyme-conjugated Avidin will exhibit a change in color. The enzyme-substrate reaction is terminated by the addition of a sulphuric acid solution and the color change is measured spectrophotometrically at a wavelength of 450 nm +/- 2 nm. The concentration of BSP in the samples is then determined by comparing the O.D. of the samples to the standard curve