Molecular Techniques and Methods

Protein Quantitation by Bradford Method

Copy Right © 2001/ Institute of Molecular Development LLC

INTRODUCTION

Quantitation of the amount of protein contained in a solution can be conveniently determined using calorimetric methods. The Bradford and the Lowry methods are the most frequently used and reliable procedures. The method of choice is the Bradford method, which is easy and rapid to complete. The Bradford method depends on quantitating the binding of a dye, Coomassie Brilliant Blue, to an unknown protein and comparing this binding to that of different amounts of a standard protein, usually bovine serum albumin (BSA). It is designed to quantify 1 to 10 ug protein. Protein determinations in the range of 10 to 100 ug may be carried out by increasing the volume of the dye solution 5-fold and using larger tubes.




MATERIALS AND SOLUTIONS

0.5 ug/ul Bovine Serum Albumin (BSA)


0.15 M NaCl


Coomassie Brilliant Blue Solution (100 ml)
Coomassie Brilliant Blue G-250 ------------------------- 10 mg
95% Ethanol -------------------------------------------- 5 ml
85% Phosphoric acid ----------------------------------- 10 ml
Deionized H2O ----------------------------------------- 85 ml
  • Filter through Whatman No.1 filter paper.
  • Store at 4oC.




    PROCEDURES

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

    Tube Number
    0.5 ug/ul BSA
    0.15 M NaCl
    Coomassie Brilliant Blue Solution
    1
    0
    100 ul
    1 ml
    2
    5 ul
    95 ul
    1 ml
    3
    10 ul
    90 ul
    1 ml
    4
    15 ul
    85 ul
    1 ml
    5
    20 ul
    80 ul
    1 ml
    6
    25 ul
    75 ul
    1 ml
    7
    30 ul
    70 ul
    1 ml
    8
    35 ul
    65 ul
    1 ml
    unknown
    5 ul
    95 ul
    1 ml


    2. Allow to stand 2 minutes at room temperature.

    3. Determine the A595 using a 1 cm pathlength microcuvette (1 ml) and make a standard curve by plotting absorbancc at 595 nm versus protein concentration.

    4. Determine the absorbance for the unknown and use the standard curve to determine the concentration of protein in the unknown. If the unknown protein concentration is too high, dilute the protein, or generate another standard curve in a higher concentration range (e.g., 10 to 100 ug).




    NOTES




    KIT INFORMATION




    REFERENCES

  • Bradford, M.M. 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein dye binding. Anal. Biochem. 72:248-254.

  • Brogdon, W.G. and Dickinson. C.M. 1983. A microassay system for measuring esterase activity and protein concentration in small samples and in high-pressure liquid chromatography eluate fractions. Anal. Biochem. 131:499-503.

  • Cadman E., Bostwick, J.R., and Eichberg, J. 1979. Determination of protein by a modified Lowry procedure in the presence of some commonly used detergents. Anal. Biochem. 96:21-23.

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

  • Peterson, G.L. 1977. A simplification of the protein assay method of Lowry et al. which is more generally applicable. Anal. Biochem. 83:3.46- 356.


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

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