Molecular Techniques and Methods

Protein Quantitation by Bicinchoninic Acid (BCA) Assay

Copy Right © 2001/ Institute of Molecular Development LLC

INTRODUCTION

Biconchoninic Acid (BCA) Assay is the modification of Lowry method. BCA assay is simple and accurate for determination of protein concentration.




MATERIALS AND SOLUTIONS

Reagent A (100 ml)
1% BCA (4,4'-Dicarboxy-2,2'-biquinoline) ---------------------------- 1 g
2% Na2CO3-H2O ----------------------------------------------------- 2 g
0.16% Sodium tartarate ------------------------------------------------ 0.16 g
0.4% NaOH ----------------------------------------------------------- 0.4 g
0.95% NaHCO3 ------------------------------------------------------- 0.95 g
Add deionized H2O to make a final volume of ------------------------ 100 ml


Reagent B (2 ml)
4% CuSO4-5H2O ----------------------------------------------------- 0.08 g
Add deionized H2O to make a final volume of ------------------------ 2 ml


Reagent C (102 ml)
Reagent A -------------------------------------------------------------- 100 ml
Reagent B -------------------------------------------------------------- 2 ml
  • Store at 4oC. Stable for 1 week.


    2 ug/ul Bovine Serum Albumin (BSA)




    PROCEDURES

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


    Tube Number
    2 ug/ul BSA
    Deionized H2O
    Reagent C
    Protein Concentration (ug/ml)
    1
    0
    100 ul
    2 ml
    0
    2
    0.5 ul
    99.5 ul
    2 ml
    10
    3
    1 ul
    99 ul
    2 ml
    20
    4
    2 ul
    98 ul
    2 ml
    40
    5
    3 ul
    97 ul
    2 ml
    60
    6
    4 ul
    96 ul
    2 ml
    80
    7
    5 ul
    95 ul
    2 ml
    100
    Unknown sample
    2 ul
    98 ul
    2 ml
    ------


    2. Incubate at 37oC for 30 minutes.

    3. Cool to room temperature.
    After cooling the sample to room temperature, absorbance continues to increase at about 2.4%/ 10 minutes.

    4. Read the absorbance at 562 nm.

    5. 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


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

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