QB3 Berkeley / Education / Lab Fundamentals Bootcamp / Bootcamp Manual / Protein Expression, Purification, and Analysis Overview / Protein Quantification: BCA Assay

Protein Quantification: BCA Assay

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Key Concepts

The bicinchoninic acid (BCA) assay is one of many ways to measure the total protein concentration of an unknown sample. 

In this assay, copper (II) [Cu2+] is reduced to copper (I) [Cu1+] in the presence of peptides in alkaline solutions. BCA can chelate (bind to) Cu1+, creating a purple color with a maximum absorbance at 562 nm. As the protein concentration increases, more of the BCA-Cu1+ complex will form and the absorbance will increase. This relationship is linear and therefore follows the Beer-Lambert law. The colorimetric detection is relative and must be compared against a protein standard of known concentration. 

The protein standards are prepared as follows (you will need to calculate the final concentrations on your own based on a 2000µg/mL stock solution):

Tube

 Volume of Diluent (µL) Volume and source of BSA (µL)

Final BSA Concentration(µg/mL)

I

 0 Stock

2000

II

 6.25 18.75 of Stock

1500

III

 16.25 16.25 of Stock

1000

IV

 8.75 8.75 of tube II dilution

750

V

 16.25 16.25 of tube III dilution

500

VI

 16.25 16.25 of tube V dilution

250

VII

 16.25 16.25 of tube VI dilution

125

VIII

 20 5.00 of tube VII dilution

25

IX

 20 0

0 = Blank

Materials

  • 96-well microplate
  • BCA Reagent A (component of the Pierce BCA Protein Assay Kit)
  • BCA Reagent B ((component of the Pierce BCA Protein Assay Kit)
  • Protein standards
  • Unknown protein samples

Procedure

  1. Add 10uL of each standard and your GFP elution “E” into a microplate well in duplicate. Each standard and sample should have its own well (see layout below).
1 2 3 4 5 6 7 8 9 10 11

12

A

 I II III IV V VI VII VIII IX

GFP“E”

 – –

B

 I II III IV V VI VII VIII IX GFP“E” – –

 

  1. Mix BCA Reagent A with BCA Reagent B at a 50:1 ratio (50:1, Reagent A:B). This is your working reagent. Be sure to make enough for at least 10 wells (see step 3).
  2. Add 200uL of working reagent to each well.
  3. Cover the plate and mix by gently tapping the edge.
  4. Incubate at 37°C for 30 minutes.
  5. Measure the absorbance at 562 nm.

Constructing a standard curve:

  1. Make a graph plotting the concentrations of your standards and the absorbances from the plate reader.
  2. Find the equation for the least-squares regression line of your data.
    1. On Microsoft Excel, right click the data on the graph and select the function “Add Trendline”
    2. Select the trendline and select “Display Equation on chart”. You may also wish to select “Display R-squared value on chart”
  3. Use the equation displayed to determine the protein concentration in your unknown sample.