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Absorbance (450 nm) Simple procedure Fast and convenient High-throughput adaptable
Prepare just the appropriate amounts for the assay.a) 1X Wash Buffer: Dilute 10X Wash Buffer 1: 9 with dH2O to obtain 1X Wash Buffer.b) 1X Diluent: Dilute 5X Wash Buffer 1: 4 with dH2O to obtain 1X Diluent.c) 1X Detector: Dilute 100X Detector 1: 99 with 1X Diluent to obtain 1X Detector.d) Substrate Solution: Equal volumes of Substrate Solutions I and II must be mixed together. Note: The diluted Detector must be used within 1 hr of preparation, Substrates I and II should be mixed within 15 min of use (Protect Substrate Solution from light).
Test Samples/Standards/QC Sample: (We recommend these be run in duplicate)a) Serum: Use a serum separator tube. Let samples clot at room temperature for 30 min before centrifugation for 20 min at 1000 x g. Assay freshly prepared serum or store serum in aliquots at - 20 °C for future use. Avoid repeated freeze/thaw cycles. Serum should be diluted in Diluent 1X. Samples containing visible precipitates must be clarified before use. As a starting point, 1/100 dilution is suggested. If samples fall outside the assay range a lower or higher dilution may be required.b) Urine: Aseptically collect the urine, voided directly into a sterile container. Assay immediately or aliquot and store at -20 °C. Avoid repeated freeze/thaw cycles. Urine should be diluted in Diluent 1X. Samples containing visible precipitates must be clarified before use. Note: Serum, Plasma, Urine or Cell Culture Supernatant have to be diluted in Diluent 1X. Samples containing visible precipitates must be clarified before use.c) QC Sample: Reconstitute human RBP4 QC Sample with 1 mL of dH2O. Mix the QC Sample to ensure complete reconstitution. Allow to sit for a minimum of 15 min. The QC Sample is ready to use-do not dilute it (refer to the C of A for current QC Sample concentration).d) Standards: Reconstitute human RBP4 Standard with 1 mL of dH2O to produce a stock solution (24 ng/mL). Mix the Stock solution to ensure complete reconstitution. Allow to sit for a minimum of 15 min. The reconstituted standard should be aliquoted and stored at -20 °C.e) Prepare 1X Diluent: Dilute 5X Diluent 1:4 with dH2O.f) Prepare Standard Curve using 2-fold serial dilutions with 1X Diluent.
a) Average the duplicate readings for each Standard, QC Sample and Test Sample.b) Generate a Standard Curve by plotting the average absorbance on the vertical (Y) axis vs. the corresponding concentration (µg /mL) on the horizontal (X) axis.c) Calculate the Test Sample RBP4 concentrations by interpolation of the Standard Curve regression curve in the form of a 4-parameter logistic equation.d) If the Test Samples were diluted, multiply the interpolated values by the dilution factor to calculate the corrected human RBP4 concentrations.