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    Biochemistry Practical

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    drp89726
    The document outlines the Hartree-Lowry method for protein estimation, a sensitive assay that utilizes copper ions and Folin reagent to measure protein concentration based on the presence of aromatic amino acids. It details the necessary materials, reagents, and step-by-step methodology for conducting the assay, including preparation of standard curves for accurate protein quantification. The procedure emphasizes maintaining specific pH levels and provides a framework for calculating protein amounts in unknown samples.

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    Biochemistry Practical

    Uploaded by

    drp89726
    2 views4 pages
    The document outlines the Hartree-Lowry method for protein estimation, a sensitive assay that utilizes copper ions and Folin reagent to measure protein concentration based on the presence of aromatic amino acids. It details the necessary materials, reagents, and step-by-step methodology for conducting the assay, including preparation of standard curves for accurate protein quantification. The procedure emphasizes maintaining specific pH levels and provides a framework for calculating protein amounts in unknown samples.

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    Biochem Manual

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    The document outlines the Hartree-Lowry method for protein estimation, a sensitive assay that utilizes copper ions and Folin reagent to measure protein concentration based on the presence of aromatic amino acids. It details the necessary materials, reagents, and step-by-step methodology for conducting the assay, including preparation of standard curves for accurate protein quantification. The procedure emphasizes maintaining specific pH levels and provides a framework for calculating protein amounts in unknown samples.

    Copyright:

    © All Rights Reserved
    Download as PDF, TXT or read online from Scribd
    Download as pdf or txt
    2 views4 pages

    Biochemistry Practical

    Uploaded by

    drp89726
    The document outlines the Hartree-Lowry method for protein estimation, a sensitive assay that utilizes copper ions and Folin reagent to measure protein concentration based on the presence of aromatic amino acids. It details the necessary materials, reagents, and step-by-step methodology for conducting the assay, including preparation of standard curves for accurate protein quantification. The procedure emphasizes maintaining specific pH levels and provides a framework for calculating protein amounts in unknown samples.

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    BT 210 BIOCHEMISTRY LAB

    Hartree-Lowry Method Of Protein Estimation


    Theory/Principle:

    The Lowry assay (1951) is an often-cited general use protein assay. For some time it was
    the method of choice for accurate protein determination for cell fractions,
    chromatography fractions, enzyme preparations, etc. The Hartree version of the Lowry
    assay, a more recent modification that uses fewer reagents, improves the sensitivity with
    some proteins, is less likely to be incompatible with some salt solutions, provides a more
    linear response, and is less likely to become saturated. .

    Under alkaline conditions the divalent copper ion forms a complex with peptide bonds in
    which it is reduced to a monovalent ion which reacts with folin reagent.
    Phospomolybdotungstate of folin reagent is reduced to heteromolybdenum blue by the
    copper-catalysed oxidation of aromatic amino-acids, tyrosine and tryptophan present in
    proteins.The intensity of the colour depends on the amount of the aromatic amino acids
    present and will thus vary for different proteins.The lowry method is sensitive to pH
    changes and therefore the pH of the assay should be maintained at 10-10.5.

    Methodology:

    a)Materials Required:

    (i) Equipments: Spectrophotometer.

    Glass or polystyrene cuvettes

    Water Bath
    (ii) Chemicals/reagents:

    • Sodium potassium tartrate.4H2O


    • Sodium carbonate
    • NaOH
    • Copper sulfate
    • Folin-ciocalteau reagent
    • Bovine serum albumin(BSA)

    (iii) Glass wares and others:

    • Test-tubes
    • Pipettes
    • Graduated cylinder

    b)Procedure:

    • REAGENT A:The reagent consists of

    2gm Sodium potassium tartarte.4H2O (7mM Na-K Tartrate)

    100gm Sodium carbonate (0.81M Sodium carbonate)

    500 ml 1N NaOH (0.5N NaOH)

    H2O upto 1Lt.The reagent can be kept for 2-3 months

    • REAGENT B:The reagent consists of:

    2 gm Sodium potassium tartrate.4H2O (70mM Na-K Tartrate)

    1gm Copper Sulfate (40mM CuSO4)


    10ml 1N NaOH

    90ml H2O.The reagent can be kept for 2-3 months

    • REAGENT C:

    1 vol Folin-Ciocalteau reagent diluted with15 vols of water

    • BSA stock solution:0.3mg/ml

    c)Assay:

    1.Prepare a series of dilutions of 0.3mg/ml bovine serum albumin in the same


    buffer containing the test sample to give concentrations of 30 to 150µg/ml.

    2. Tube 1 is used as blank and tubes 2 through 6 are for standard calibration curve
    for protein. Tubes7-10 are for unknown samples (Table1).To 1 ml of blank,each
    dilution of standard and test sample and add 0.9 ml Reagent A in separate tubes
    and mix.

    3.Incubate the tubes at 50ºC for 10min and cool to room temperature

    4.Add 0.1ml reagent B to each tube, mix and incubate for 10min at room
    temperature.

    5.Rapidly add 3 ml Reagent C to each tube, mix, incubate at 50ºC for10min and
    cool to room temperature.

    6.Incubate the tubes for 30-60 mins at room temperature. Measure absorbance at
    650 nm using UV-Vis spectrophotometer.
    Table1:

    Sl PROTEIN DH2O ReagentA ReagentB ReagentC A650

    (µl) (µg)
    No (µl) (ml) (ml) (ml) (nm)

    1 - - 1000 0.9 0.1 3


    2. 100 30 900 0.9 0.1 3
    3. 200 60 800 0.9 0.1 3
    4 300 90 700 0.9 0.1 3
    5. 400 120 600 0.9 0.1 3
    6. 500 150 500 0.9 0.1 3
    7. 1000 - - 0.9 0.1 3
    8. Unknown - - 0.9 0.1 3
    Sample (A)

    9. Unknown - - 0.9 0.1 3


    Sample (B)
    10. Unknown - - 0.9 0.1 3
    Sample (C)

    Calculation: Prepare a standard curve of absorbance versus micrograms protein and


    determine the slope y/x from the standard curve, which gives the A650 per
    unit of protein(µg).Hence determine the amount of protein in the unknown
    sample.

    References:

    1.Lowry,OH,NJ Rosbrough,AL Farr,and RJ Randall.(1951).J.Biol.Chem.193:265.

    2.Hartree,EF.(1972)Anal Biochem.48:422-427

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