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    Home > Biochemistry News > Biotechnology News > Phosphate Assay by Suprya Jaydev

    Phosphate Assay by Suprya Jaydev

    • Last Update: 2020-11-22
    • Source: Internet
    • Author: User
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    Phosphate Assay by Suprya Jaydev
    Contributor: Suprya Jayadev
    Date: Sep. 8, 1994
    ReagentsAshing buffer:
    10 g Mg(NO3)2
    100 ml EtOH
    1.5 N HCl stock:
    119.7 ml concentrated HCl (11.6M @ 36% by weight)
    880.3 ml H2O
    1 N Sulfuric acid stock:
    28.9 ml concentrated sulfuric acid (18M/36N @ 96% by weight)
    971.1 ml H2O
    Ammonium molybdate stock:
    4.2 ml Ammonium molybdate
    1000 ml 1N H2SO4
    Procedure
    1) Prepare standards in duplicate.
    --> standards: 0, 3, 5, 7, 10, 12, and 15 µl of a 1 mM NaH2PO4 stock solution
    2) Aliquot lipid unknowns.
    3) Add 100 µl ashing buffer to each sample.
    4) Dry EtOH and chloroform from samples by heating @ 80°C for ~20 minutes.
    5) Flame samples using a strong flame on the bunsen burner.
    6) Remove samples from flame when the brown gas ceases to be released.
    --> There should be a white percipitate remaining at the base of the tubes. If the ashing process is allowed to proceed for too long, the percipitate will appear brown/charred and this will skew the spectrophotometer readings.
    7) Add 0.3 ml of 0.5N HCl to each sample and boil for 15 minutes.
    8) To each tube, add:
    0.6ml ammonium molybdate
    0.1 ml ascorbic acid (10% w/v, made fresh EACH time)
    VORTEX!!
    9) Incubate for 30 minutes @ 45°C or for 60 minutes @ 37°C.
    10) Read OD820.
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