Molecular Techniques and Methods

Protein Quantitation by Lowry Method

Copy Right © 2001/ Institute of Molecular Development LLC

INTRODUCTION

The most accurate method of determining protein concentration is acid hydrolysis followed by amino acid analysis. Most other methods are sensitive to the amino acid composition of the protein and absolute concentrations cannot be obtained. The procedure of Lowry et. al. is no exception, but its sensitivity is moderately constant from protein to protein, and it has been so widely used.




MATERIALS AND SOLUTIONS

Complex-Forming Reagent
  • Solution A: 2% (w/v) Na2CO3 in distilled water
  • Solution B: 1% (w/v) CuS04-5H2O in distilled water
  • Solution C: 2% (w/v) Sodium Potassium Titrate in distilled water
  • Prepare immediately before use by mixing Solution A: Solution B: Solution C = 100: 1: 1 (v/v/v).


    2N NaOH


    Folin-Ciocalteu Reagent (commercially available)
    Use at 1 N concentration.


    Bovine Serum Albumin (Fraction V)
    BSA ------------------------------------------ 4 mg
    Deionized H2O ------------------------------- 1 ml
  • Store frozen at - 20oC.




    PROCEDURES

    1. Mix the following components into microfuge tubes and make duplicate tubes.

    Tube Number
    4 ug/ul BSA
    Deionized H2O
    2 N NaOH
    Protein Concentration (ug/ml)
    1
    0
    100 ul
    100 ul
    0
    2
    0.25 ul
    99.75 ul
    100 ul
    10
    3
    0.5 ul
    99.5 ul
    100 ul
    20
    4
    1.25 ul
    98.75 ul
    100 ul
    50
    5
    2.5 ul
    97.5 ul
    100 ul
    100
    6
    5 ul
    95 ul
    100 ul
    200
    7
    12.5 ul
    87.5 ul
    100 ul
    500
    8
    25 ul
    75 ul
    100 ul
    1,000
    9
    50 ul
    50 ul
    100 ul
    2,000
    Unknown
    50 ul
    50 ul
    100 ul
    ------


    2. Hydrolyze protein at 100oC for 10 minutes.

    3. Cool the hydrolyzate to room temperature.

    4. Add 1 ml of freshly mixed Complex-Forming Reagent.

    5. Let the solution stand at room temperature for 10 minutes.

    6. Add 0.1 ml of Folin-Ciocalteu reagent, and vortex to mix.

    7. Incubate at room temperature for 30-60 minutes (do not exceed 60 minutes).

    8. If the protein concentration was below 500 ug/ ml, read the absorbance at 750 nm.
    If the protein concentration was between 100 and 2,000 ug/ml, read the absorbance at 550 nm.

    9. Plot a standard curve of absorbance as a function of initial protein concentration and use it to determine the unknown protein concentrations.




    NOTES




    KIT INFORMATION




    REFERENCES

  • Lowry, O.H., Rosebrough, N.J., Farr, A.L., and Randall, R.J., 1951, Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193, 265-275.


  • Please send your comment on this protocol to "editor@MolecularInfo.com".

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