Phosphoenolpyruvate Carboxykinase (PEPCK) BioAssay™ ELISA Kit (Rat)

For quantitative detection of PEPCK in serum, plasma, tissue homogenates and other biological fluids.||Detection Range:|15.625-1000pg/ml||Sensitivity:|9.375pg/ml||Precision: |Intra-Assay %CV: <8%|Inter-Assay %CV: <10%||Specificity:|This assay has high sensitivity and excellent specificity for detection of PEPCK. No significant cross-reactivity or interference between PEPCK and analogues was observed.||Assay Principle:|This BioAssay™ kit was based on sandwich enzyme-linked immune-sorbent assay technology. Capture antibody was pre-coated onto 96-well plates. And the biotin conjugated antibody was used as detection antibodies. The standards, test samples and biotin conjugated detection antibody were added to the wells subsequently, and washed with wash buffer. HRP-Streptavidin was added and unbound conjugates were washed away with wash buffer. TMB substrates were used to visualize HRP enzymatic reaction. TMB was catalyzed by HRP to produce a blue color product that changed into yellow after adding acidic stop solution. The density of yellow is proportional to the target amount of sample captured in plate. Read the O.D. absorbance at 450nm in a microplate reader, and then the concentration of target can be calculated.||Kit Components:|Microtiter Strips, 1x96 wells|Standard, 2x1 vial|Sample/Standard Dilution Buffer, 1x20ml|Antibody (Biotin) (Concentrated), 1x60ul|Antibody Dilution Buffer, 1x10ml|Streptavidin (HRP) (SABC), 1x120ul|SABC Dilution Buffer, 1x10ml|TMB Substrate, 1x10ml|Wash Buffer, 25X, 1x30ml||Storage and Stability:|Store unopened Microtiter Strips at 4ºC; store at -20ºC once opened. Store unopened Standard at 4°C; once reconstituted, store at 4°C for up to 12 hours or at -20°C for up to 48 hours. Store other components at 4°C. Kit is stable for 6 months after receipt. For maximum recovery of product, centrifuge the original vials after thawing and prior to removing the cap.||Assay Summary:|1. Wash plate 2 times before adding standards and samples to wells!|2. Add 50ul standard or sample to each well.|3. Add 50ul Antibody (Biotin) working solution to each well and incubate for 45 minutes at 37°C|4. Aspirate and wash 3 times.|5. Add 100ul SABC working solution to each well. Incubate for 30 minutes at 37°C|6. Aspirate and wash 5 times.|7. Add 90ul TMB substrate. Incubate 10-20 minutes at 37°C|8. Add 50ul Stop Solution (Customer supplied). Read at 450nm immediately.|9. Calculate results