SPARC ELISA Kit (Rat)

Intra-assay Precision (Precision within an assay) : 3 samples with low, middle and high level Osteonectin (ON) were tested 20 times on one plate, respectively. Inter-assay Precision (Precision between assays) : 3 samples with low, middle and high level Osteonectin (ON) were tested on 3 different plates, 8 replicates in each plate. CV (%) = SD/meanX100 Intra-Assay: CV<10% Inter-Assay: CV<12%

Basement-membrane protein 40; BM-40; ON; osteonectin; secreted acidic cysteine rich glycoprotein; Secreted acidic cystein-rich glycoprotein (osteonectin) ; secreted protein acidic and rich in cysteine; secreted protein, acidic, cysteine-rich (osteonectin) ; SPARC.

Appears to regulate cell growth through interactions with the extracellular matrix and cytokines. Binds calcium and copper, several types of collagen, albumin, thrombospondin, PDGF and cell membranes. There are two calcium binding sites; an acidic domain that binds 5 to 8 Ca (2+) with a low affinity and an EF-hand loop that binds a Ca (2+) ion with a high affinity.

The test principle applied in this kit is Sandwich enzyme immunoassay. The microtiter plate provided in this kit has been pre-coated with an antibody specific to Osteonectin (ON) . Standards or samples are then added to the appropriate microtiter plate wells with a biotin-conjugated antibody specific to Osteonectin (ON) . Next, Avidin conjugated to Horseradish Peroxidase (HRP) is added to each microplate well and incubated. After TMB substrate solution is added, only those wells that contain Osteonectin (ON), biotin-conjugated antibody and enzyme-conjugated Avidin will exhibit a change in color. The enzyme-substrate reaction is terminated by the addition of sulphuric acid solution and the color change is measured spectrophotometrically at a wavelength of 450nm +/- 10nm. The concentration of Osteonectin (ON) in the samples is then determined by comparing the O.D. of the samples to the standard curve.

Reconstitution & Storage Instructions Western Blotting/Immunoblotting (WB/IB) Protocol Immunohistochemistry (IHC) Protocol Immunocytochemistry (ICC) Protocol Enzyme-Linked ImmunoSorbent Assay (ELISA) Protocol Blocking Peptide Competition Protocol (BPCP) Immunoprecipitation (IP) Protocol Antibody Array (AA) Protocol Reconstitution & Storage Instructions Reconstitution & Storage Instructions Western Blotting/Immunoblotting (WB/IB) Protocol Western Blotting/Immunoblotting (WB/IB) Protocol Immunohistochemistry (IHC) Protocol Immunohistochemistry (IHC) Protocol Immunocytochemistry (ICC) Protocol Immunocytochemistry (ICC) Protocol Enzyme-Linked ImmunoSorbent Assay (ELISA) Protocol Enzyme-Linked ImmunoSorbent Assay (ELISA) Protocol Blocking Peptide Competition Protocol (BPCP) Blocking Peptide Competition Protocol (BPCP) Immunoprecipitation (IP) Protocol Immunoprecipitation (IP) Protocol Antibody Array (AA) Protocol Antibody Array (AA) Protocol

Matrix Recovery Range (%) Average (%) serum (n=5) 79-90 83 EDTA plasma (n=5) 78-105 91 heparin plasma (n=5) 86-95 91 Matrix Recovery Range (%) Average (%) Matrix Recovery Range (%) Average (%) serum (n=5) 79-90 83 serum (n=5) 79-90 83 EDTA plasma (n=5) 78-105 91 EDTA plasma (n=5) 78-105 91 heparin plasma (n=5) 86-95 91 heparin plasma (n=5) 86-95 91

Component Amount Anti-Sparc Microplate 96 Wells (12 x 8 Well Strips) Sparc Lyophilized Standard 2 x 40 ng 100X Biotinylated Sparc Detector Antibody 120 uL Avidin/HRP Conjugate 120 uL Standard Diluent 1 x 20 mL Detector Antibody Diluent 1 x 12 mL Conjugate Diluent 1 x 12 mL 30X Wash Buffer 1 x 20 mL TMB Substrate 1 x 9 mL Stop Solution 1 x 6 mL Component Amount Component Amount Anti-Sparc Microplate 96 Wells (12 x 8 Well Strips) Anti-Sparc Microplate 96 Wells (12 x 8 Well Strips) Sparc Lyophilized Standard 2 x 40 ng Sparc Lyophilized Standard 2 x 40 ng 100X Biotinylated Sparc Detector Antibody 120 uL 100X Biotinylated Sparc Detector Antibody 120 uL Avidin/HRP Conjugate 120 uL Avidin/HRP Conjugate 120 uL Standard Diluent 1 x 20 mL Standard Diluent 1 x 20 mL Detector Antibody Diluent 1 x 12 mL Detector Antibody Diluent 1 x 12 mL Conjugate Diluent 1 x 12 mL Conjugate Diluent 1 x 12 mL 30X Wash Buffer 1 x 20 mL 30X Wash Buffer 1 x 20 mL TMB Substrate 1 x 9 mL TMB Substrate 1 x 9 mL Stop Solution 1 x 6 mL Stop Solution 1 x 6 mL

This assay has high sensitivity and excellent specificity for detection of Osteonectin (ON) . No significant cross-reactivity or interference between Osteonectin (ON) and analogues was observed.