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    Biuret Method

    Uploaded by

    Larry Luciano
    The document describes the Biuret method for determining protein concentration. It involves mixing a protein sample with an alkaline copper solution, which forms a purple complex between copper and peptide bonds. The concentration of the complex is determined colorimetrically and used to calculate the protein concentration in the original sample by comparing to a standard curve of known protein concentrations. Reagents, equipment, procedures, calculations, and references are provided to carry out the assay.

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    © All Rights Reserved
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    Biuret Method

    Uploaded by

    Larry Luciano
    57 views2 pages
    The document describes the Biuret method for determining protein concentration. It involves mixing a protein sample with an alkaline copper solution, which forms a purple complex between copper and peptide bonds. The concentration of the complex is determined colorimetrically and used to calculate the protein concentration in the original sample by comparing to a standard curve of known protein concentrations. Reagents, equipment, procedures, calculations, and references are provided to carry out the assay.

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    The document describes the Biuret method for determining protein concentration. It involves mixing a protein sample with an alkaline copper solution, which forms a purple complex between copper and peptide bonds. The concentration of the complex is determined colorimetrically and used to calculate the protein concentration in the original sample by comparing to a standard curve of known protein concentrations. Reagents, equipment, procedures, calculations, and references are provided to carry out the assay.

    Copyright:

    © All Rights Reserved
    Download as PDF, TXT or read online from Scribd
    Download as pdf or txt
    57 views2 pages

    Biuret Method

    Uploaded by

    Larry Luciano
    The document describes the Biuret method for determining protein concentration. It involves mixing a protein sample with an alkaline copper solution, which forms a purple complex between copper and peptide bonds. The concentration of the complex is determined colorimetrically and used to calculate the protein concentration in the original sample by comparing to a standard curve of known protein concentrations. Reagents, equipment, procedures, calculations, and references are provided to carry out the assay.

    Copyright:

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

    PROTEIN DETERMINATION

    Biuret Method

    PRINCIPLE:
    Alkaline pH
    Copper + Protein > Copper-protein complexes

    CONDITIONS: T = 25°C, A540nm, Light path = 1 cm

    METHOD: Colorimetric

    REAGENTS:

    A. 0.85% (w/v) Sodium Chloride Solution (NaCl)


    (Use Sodium Chloride Solution, 0.85%, Sigma Stock No. 430AG-4 or prepare 100 ml in
    deionized water using Sodium Chloride, Sigma Prod. No. S-9625.)

    B. 0.5% (w/v) Working Protein Standard (WPS)


    (Use WPS prepared per Sigma "Preparation of Working Protein Standard for use in Protein
    Determination Procedure." (See attached procedure)

    C. Biuret Reagent (Biuret)


    (Use Total Protein Reagent, Sigma Stock No. 541-2.)

    D. Protein Sample Solution (Protein)


    (Prepare a solution containing 0.5 - 4 mg protein/ml of Sample in Reagent A.)

    PROCEDURE:

    Pipette (in milliliters) the following reagents into suitable containers:

    Test Std 1 Std 2 Std 3 Std 4 Std 5 Blank

    Reagent A (NaCl) ---- 0.96 0.90 0.80 0.40 ---- 1.00


    Reagent B (WPS) ---- 0.04 0.10 0.20 0.60 1.00 ----
    Reagent D (Protein) 1.00 ---- ---- ---- ---- ---- ----

    Mix by swirling. Then add:

    Reagent C (Biuret) 4.00 4.00 4.00 4.00 4.00 4.00 4.00

    Mix thoroughly by vortexing and incubate for 30 minutes at 25°C. Transfer to suitable cuvettes and
    record the absorbance at 540 nm for Test, Standards, and Blank.

    SSPBIU01.001 Page 1 of 2
    Revised: 05/16/95
    PROTEIN DETERMINATION
    Biuret Method

    CALCULATIONS:

    r A 540nm Standard = A540nm Std - A540nm Std Blank

    Prepare a standard curve by plotting the A540nm of the Standards vs mg of protein.

    Sample Determination:

    r A 540nm Sample = r A540nm Test - A540nm Test Blank

    Determine the mg of protein using the Standard curve.

    mg Protein = (mg of protein from the Standard curve)(df)

    (mg Protein) (100)


    ___________________________
    % Protein =
    (mg solid/ml Reagent D)

    For Products that are liquid:

    (mg Protein)
    _________________
    mg Protein/ml =
    (ml Reagent D)

    100 = Conversion to percentage

    REFERENCES:

    Gornall, A.G., Bardawill, C.J. and David, M.M. (1949) J. Biol. Chem. 177, 751-766

    Ryan, M.T. and Chopra, R.K. (1976) Biochim. Biophys. Acta 427, 337-349

    NOTES:

    1. This assay is based on the cited references.

    2. Where Sigma Product or Stock numbers are specified, equivalent reagents may be
    substituted.

    This procedure is for informational purposes. For a current copy of Sigma’s quality control
    procedure contact our Technical Service Department.

    SSPBIU01.001 Page 2 of 2
    Revised: 05/16/95

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