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Protein determination by the BCA method
 

 

 
 

 

 

Absorbance assays: [absorbance at 280 nm] [absorbance at 205 nm] [extinction coefficient]
Colorimetric assays: [modified Lowry] [biuret] [Bradford] [Bicinchoninic Acid (Smith)]
Methods: [set up a standard curve] [spectrophotometry]

Bicinchoninic Acid (BCA) Protein Assay (Smith)

Considerations for use

The bicinchoninic acid (BCA) assay is available in kit form from Pierce (Rockford, Ill.). This procedure is very applicable to microtiter plate methods. The BCA is used for the same reasons the Lowry is used. Stoscheck (1990) has suggested that the BCA assay will replace the Lowry because it requires a single step, and the color reagent is stable under alkaline conditions.

Both a standard assay for concentrated proteins and a micro assay for dilute protein solutions are described below.

Principle

BCA serves the purpose of the Folin reagent in the Lowry assay, namely to react with complexes between copper ions and peptide bonds to produce a purple end product. The advantage of BCA is that the reagent is fairly stable under alkaline conditions, and can be included in the copper solution to allow a one step procedure. A molybdenum/tungsten blue product is produced as with the Lowry.

Equipment

In addition to standard liquid handling supplies a visible light spectrophotometer is needed with transmission set to 562 nm. Glass or polystyrene (cheap) cuvettes may be used.

Procedure 1 (standard assay)

Reagents

  1. Reagent A: 1 gm sodium bicinchoninate (BCA), 2 gm sodium carbonate, 0.16 gm sodium tartrate, 0.4 gm NaOH, and 0.95 gm sodium bicarbonate, brought to 100 ml with distilled water. Adjust the pH to 11.25 with 10 M NaOH.
  2. Reagent B: 0.4 gm cupric sulfate (5 x hydrated) in 10 ml distilled water.
  3. Standard working solution (SWR): Mix 100 volumes reagent A with 2 volumes reagent B.
  4. The stock solutions are stable. The working solution is stable for 1 week and should be green.

Assay

  1. Prepare samples containing 0.2 to 50 micrograms protein in microliters.
  2. Add 1 ml SWR to each 20 microliters sample and mix. Incubate 30 min. at 60 degrees C.
  3. Cool the samples and read at 562 nm. Color will be stable for at least one hour.

Procedure 2 (micro assay)

Reagents

  1. Reagent A: 8 gm sodium carbonate monohydrate, 1.6 gm sodium tartrate, brought to 100 ml with distilled water. Adjust the pH to 11.25 with 10 M NaOH.
  2. Reagent B: 4 gm BCA in 100 ml distilled water.
  3. Reagent C: 0.4 gm cupric sulfate (5 x hydrated) in 10 ml water.
  4. Working solution: Mix 1 volume reagent C with 25 volumes reagent B, then add 26 volumes reagent A to the C/B mixture.

Assay

  1. Prepare samples containing 0.2 to 50 micrograms protein in 500 microliters.
  2. Add 500 microliters working solution to each 500 microliters sample and mix. Incubate 60 min. at 60 degrees C.
  3. Cool the samples and read at 562 nm.

Analysis

Prepare a standard curve of absorbance versus micrograms protein (or vice versa), and determine amounts from the curve. Determine concentrations of original samples from the amount protein, volume/sample, and dilution factor, if any. If you are unfamiliar with how to obtain a protein concentration for a diluted sample from a standard curve, see how to prepare and use a protein standard curve.

Comments

A longer incubation increases the sensitivity of the assay. The heating can be stopped earlier to prevent the color from becoming too dark. The assay can be performed at room temperature, but there is greater variability among proteins and the assay is less sensitive.

Reference

Stoscheck, CM. Quantitation of Protein. Methods in Enzymology 182: 50-69 (1990).

 

 


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Created by David R. Caprette (caprette@rice.edu), Rice University 24 May 95
Updated 19 May 05