& Data Analysis
Protein gel analysis
|List of methods
Direct absorbance measurement
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.
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.
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)
Procedure 2 (micro assay)
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.
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.
Stoscheck, CM. Quantitation of Protein. Methods in Enzymology 182: 50-69 (1990).