Iron Assay Kit, BioAssay(TM)

Iron level in blood is a reliable diagnostic indicator of various disease states. Increased levels of iron concentration in blood are associated with blood loss, increased destruction of red blood cells (e.g. hemorrhage) or decreased blood cell survival, acute hepatitis, certain sideroachrestic anemias, ingestion of iron-rich diets, defects in iron storage (e.g. pernicious anemia). Decreased levels of blood iron may result from insufficient iron ingestion from diets, chronic blood loss pathologies, or increased demand on iron storage as during normal pregnancy.\n\nSimple, direct and automation-ready procedures for measuring iron concentrations find wide applications in research, drug discovery and environmental monitoring. Iron assay kit is designed to measure total iron directly in serum without any pretreatment. The improved method utilizes a chromogen that forms a blue colored complex specifically with Fe2+. Fe3+ in the sample is reduced to Fe2+, thus allowing the assay for total iron concentration. The intensity of the color, measured at 590nm, is directly proportional to the iron concentration in the sample.\n\nKey Features:\nSensitive and accurate. Linear detection range 27ug/dL (4.8uM) to 1,000ug/dL (179uM) iron in 96-well plate assay.\nSimple and high-throughput. The procedure involves addition of a single working reagent and incubation for 40 min. Can be readily automated as a high-throughput assay for thousands of samples per day.\nImproved reagent stability and versatility. The optimized formulation has greatly enhanced reagent and signal stability. Cuvette or 96-well plate assay.\nLow interference in biological samples. No pretreatments are needed. Assays can be directly performed on serum samples.\n\nApplications:\nDirect Assays: iron in biological samples (e.g. serum).\nDrug Discovery/Pharmacology: effects of drugs on iron metabolism.\nEnvironmental Monitoring: iron in soil extracts, mineralized samples.\n\nKit Contents: (250 tests in 96-well plates)\nReagent A: 50ml Reagent B: 4ml Reagent C: 4ml\nIron Standard: 1ml 10 mg/dL Fe2+\nStorage conditions. The kit is shipped at room temperature. Store Reagent A at room temperature and other reagents at 4 degrees C. Shelf life: 12 months after receipt.\nPrecautions: reagents are for research use only. Normal precautions for laboratory reagents should be exercised while using the reagents. Please refer to Material Safety Data Sheet for detailed information.\n\nProcedures:\nNote: (1). Iron chelators (e.g. EDTA) interfere with this assay and should be avoided in sample preparation. (2). Serum or plasma samples should be clear and free of precipitates or turbidity. If not, centrifuge or filter to clarify samples prior to assay. (3).This kit can be applied to measure Fe2+ (vs. total iron) content. Prepare Working Reagent by mixing 20 vol of Reagent A, 1 vol of water and 1 vol Reagent C (no reductant in the Working Reagent). The procedure is the same as described for the total iron assay.\nProcedure using 96-well plate:\n1. Standards. Prepare 400ul 1000 ug/dL Premix by mixing 40ul 10mg/dL standard and 360ul distilled water. Dilute standards as follows. \nNo Premix+H2O Vol (ul) Fe (ug/dL)\n1 100ul+0ul 100 1000\n2 80ul+20ul 100 800\n3 60ul+40ul 100 600\n4 40ul+60ul 100 400\n5 30ul+70ul 100 300\n6 20ul+80ul 100 200\n7 10ul+90ul 100 100\n8 0ul+100ul 100 0\nTransfer 50ul diluted standards and 50ul sample into a clear flat bottom 96-well plate. For serum/plasma samples, it is recommended to run a sample blank (i.e. a 50ul sample in a separate well).\n2. Prepare enough Working Reagent by mixing 20 volumes of Reagent A, 1 volume Reagent B and 1 volume Reagent C. Fresh reconstitution is recommended. Equilibrate to room temperature before assay. Add 200ul Working Reagent to Standards and Samples wells. (For serum/plasma samples which require a Sample Blank Control, add 200ul Reagent A to the Sample Blank wells). Tap plate to mix.\n3. Incubate 40 min at room temperature and read optical density at 510-630nm (peak absorbance at 590nm).\nProcedure using cuvette:\n1. Prepare standards as in 96-well assay. Set up centrifuge tubes labeled Standards and Samples. Transfer 250ul standards and samples to tubes.\n2. Add 1000ul Working Reagent to all tubes. Mix by vortexing. Incubate 40 min at room temperature.\n3. Transfer to cuvettes and read OD at 590nm (510nm-630nm).\n\nCalculation:\nSubtract OD of пїЅ0 ug/dL FeпїЅ from all other standard OD values and plot the OD against standard iron concentrations. Determine the slope using linear regression fitting. Iron concentration of the sample is calculated as\n[Iron]=ODSAMPLE-ODBLANK\nSlope (ug/dL)\nWhere ODBLANK is OD values of the water blank (Standard #8), or Sample Blank, if a sample blank is used (e.g. serum or plasma). Typical serum iron values: 70-180 ug/dL.\nConversions: 1 mg/dL Fe equals 179uM, 0.001% or 10 ppm.\n\nMaterials Required, But Not Provided:\nPipeting devices and accessories.\nProcedure using 96-well plate:\nClear bottom 96-well plates (e.g. Corning Costar) and plate reader.\nProcedure using cuvette:\nCuvets and spectrophotometer for measuring OD at 510-630nm.\n\nExamples:\nMouse serum, fetal bovine serum, and goat serum were assayed using the 96-well plate assay protocol. The iron concentrations were 173 пїЅ 2 (n=4), 149 пїЅ 1 (n=4), 88 пїЅ 2 microg/dL (n=4), respectively. Coefficient of variance < 2%.

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