Magnesium (Mg) is one of the most abundant and essential minerals in mammals. Magnesium is involved in more than 300 biochemical reactions in the body and plays important roles in muscle and nerve functions, heart rhythm, immune system and bone formation. Magnesium deficiency may lead to nausea, fatigue, muscle contractions, hypocalcemia and hypokalemia.||Simple, direct and automation-ready procedures for measuring magnesium concentration in biological samples are becoming popular in Research and Drug Discovery. Magnesium assay kit is designed to measure magnesium directly in biological samples without any pretreatment. A calmagite dye in the kit forms a colored complex specifically with magnesium. The intensity of the color, measured at 500 nm, is directly proportional to the magnesium concentration in the sample. The optimized formulation minimizes interference by potential substances.||Key Features:|Sensitive and accurate. Use as little as 5ul sample. Linear detection range 0.1 mg/dL (41uM) to 3 mg/dL (1.2mM) Mg2+ in 96-well plate assay.|Simple and high-throughput. The procedure involves addition of two reagents and measuring OD500nm. Can be readily automated as a highthroughput assay for thousands of samples per day.|Improved reagent stability and versatility. The optimized formulation has greatly enhanced reagent and signal stability. Cuvet or 96-well plate assay.|Low interference in biological samples. Assays can be directly|performed in serum and urine samples.||Applications:|Direct Assays: Mg2+ in serum, urine and deproteinated samples (e.g. milk) etc.|Drug Discovery/Pharmacology: effects of drugs on Mg2+ metabolism.|Food and Beverages: Mg2+ determination.|Environment: Mg2+ determination in water and soil.||Kit Contents: (250 tests in 96-well plates)|Reagent A: 25ml Reagent B: 25ml|EDTA Solution: 2 x 1.5ml Standard: 1ml 10 mg/dL Mg2+|Storage conditions. The kit is shipped at room temperature. Store Reagent and Standard at 4°C. Shelf life: 12 months after receipt. Precautions: 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.||Procedures:|Reagent Preparation: Prepare enough working reagent by combining equal volumes of Reagent A and Reagent B. Equilibrate to room temperature before use.|Procedure using 96-well plate:|1. Dilute Standard to 2 mg/dL by mixing 40ul 10 mg/dL Standard with 160ul distilled water. Transfer 5ul diluted standard and samples in duplicate to wells of a clear bottom plate. Diluted standard can be stored at 4°C for future use.|2. Add 200ul working reagent and tap plate to mix thoroughly.|3. Incubate 2 min at room temperature and read optical density at 500 nm (OD for sample and standard).|4. Add 10ul EDTA Solution to all sample wells and tap plate to mix thoroughly. Incubate 2 min and read OD at 500nm (OD for blanks).|Procedure using cuvette:|1. Set up test tubes and transfer 25ul diluted Standard and samples to appropriately labeled tubes.|2. Add 1000ul working reagent and vortex to mix. Incubate 2 min. Transfer to cuvet and read OD500nm. Add 50ul EDTA solution, mix well, incubate 2 min and read OD500 nm.||Calculation:|Magnesium concentration of the sample is calculated as|=|ODSAMPLE–ODBLANK|ODMG-ODMGBlank|X 2 (mg/dL)|ODSAMPLE and ODBLANK are OD500nm values of sample before and after the addition of EDTA. ODMG and ODMGBLANK are OD500nm values of the standard (2 mg/dL) before and after the addition of EDTA.|Conversions: 1 mg/dL Mg2+ equals 411uM, 0.001% or 10 ppm.||Materials Required, But Not Provided:|Pipeting devices and accessories (e.g. 5ul).|Procedure using 96-well plate:|Clear bottom 96-well plates (e.g. Corning Costar) and plate reader.|Procedure using cuvette:|Cuvets and spectrophotometer for measuring OD500nm.||General Considerations:|1. EDTA and other Mg2+ chelators interfere with this assay. This assay can not be applied to plasma samples obtained with EDTA.|2. Sample pretreatment: for milk and other lipid/protein-rich samples, mix equal volumes of sample and 10% trichloroacetic acid. Incubate 5 min at room temperature and pellet precipitates for 2 min at 14,000 rpm in a table centrifuge. Assay the supernatant (dilution factor=2) using the above procedure.||Examples:|Samples were assayed in duplicate using the 96-well plate protocol. The Mg2+ values (mg/dL) were 1.64 ± 0.04 (rat serum), 1.77 ± 0.02 (human serum), 2.41 ± 0.5 (goat serum), 2.80 ± 0.14 (Invitrogen fetal bovine serum).