QuantiChrom™ Phosphate Assay Kit 500 tests

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    DESCRIPTION 

    Phosphate (Pi) is one of the most important ion species in nature. Phosphate is present in all biological systems. It is a major constituent in minerals and fertilizers, and is a component of industrial wastewater. Thus accurate determination of phosphate concentration finds numerous applications in pharmacology, biomedical research, clinical chemistry, industrial process monitoring and environmental monitoring. 

    Simple, direct and automation-ready procedures for measuring phosphate concentration in biological and environmental samples are becoming popular. BioAssay Systems' phosphate assay kit is designed to measure phosphate ion directly in samples without any pretreatment. The improved Malachite Green method utilizes the malachite green dye and molybdate, which forms a stable colored complex specifically with inorganic phosphate. The intensity of the color, measured at 620nm, is directly proportional to the phosphate concentration in the sample. The optimized formulation substantially reduces interference by substances in the raw samples.

    KEY FEATURES 

    Sensitive and accurate. Linear detection range 0.30 µM (0.0028 mg/dL) to 50 µM (0.47 mg/dL) phosphate in 96-well plate assay. 

    Simple and high-throughput. The procedure involves addition of a single working reagent and incubation for 30 min. Can be readily automated as a high-throughput assay for thousands of samples per day. 

    Improved reagent stability and versatility. The optimized formulation has greatly enhanced reagent and signal stability. Assays can be executed in cuvet or 96-well plate. Low interference in biological samples. No pretreatments are needed. Assays can be directly performed on raw biological samples i.e., in the presence of lipid, protein and minerals.  

    APPLICATIONS 

    Direct Assays: Pi in serum, urine, saliva, sweat, tissue culture etc. 

    Drug Discovery/Pharmacology: effects of drugs on Pi metabolism. 

    Food and Beverages: Pi determination. Environment: Pi determination in water, soil and fertilizer.

    KIT CONTENTS (500 tests in 96-well plates) 

    ​Reagent: 50 mL ​Pi standard: 14 mL 0.28 mg/dL (30 µM) 

    ​Blank Control: 14 mL

     Storage conditions. The kit is shipped at room temperature. Store all components 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: 

    Important: bring reagents to room temperature and shake before use. 

    Procedure using 96-well plate: 

    1. Set up standards and samples. Transfer 50 µL Blank Control, Standard and samples separately in duplicate wells of a clear bottom 96-well plate. 

    2. Add 100 µL Reagent and tap lightly to mix. 

    3. Incubate 30 min at room temperature and read optical density at 620nm (600-660nm).

    Procedure using cuvette: 

    1. Set up test tubes labeled Blank, Standard, Samples. Transfer 400 µL Blank Control, Standard and Samples to appropriately labeled tubes. 

    2. Add 800 µL Reagent and tap lightly to mix. 

    3. Incubate 30 min at room temperature, transfer to cuvet and read optical density at 620 nm (600-660nm). 

    Important: (1) if sample phosphate concentration is higher than 50 µM, dilute samples in distilled water and repeat the assay. (2) It is not necessary to prepare a calibration curve, because the concentration of the provided standard lies within the linear range. (3) Precipitation may occur at high concentrations of phosphate (>100 µM), or in the presence of high concentrations of e.g. proteins and metals. In this case, dilute samples in distilled water and repeat the assay.

    CALCULATION 

    The phosphate concentration of Sample is calculated as

     

    ODBLANK, ODSTANDARD and ODSAMPLE are OD620nm values of Blank, Standard and Sample, respectively. Conversions: 1 mg/dL Pi equals 105.3 µM, 0.001% or 10 ppm

    MATERIALS REQUIRED, BUT NOT PROVIDED 

    Pipeting devices and accessories. 

    Procedure using 96-well plate:

    Clear bottom 96-well plates (e.g. Corning Costar) and plate reader. 

    Procedure using cuvette: 

    Spectrophotometer and cuvets for measuring OD 620nm.  

    EXAMPLES (96-well plate assay):

    PUBLICATIONS 

    1. Abranches, J. (2008). CcpA regulates central metabolism and virulence gene expression in Streptococcus mutans. J Bacteriol. 190(7):2340-9. 

    2. Hildebrand, J. et al (2009) Functional and energetic characterization of P-gp-mediated doxorubicin transport in rainbow trout. Comp Biochem Physiol C Toxicol Pharmacol. 149(1):65-72. 

    3. Dunbar, D.R. et al. (2010). Transcriptional and physiological responses to chronic ACTH treatment by the mouse kidney. Physiol Genomics 40(3): 158-166.