Bicinchoninic acid (BCA) assay in low volume

Area of Science:

• Biochemistry
• Analytical Chemistry
• Biotechnology

Background:

• The Bicinchoninic Acid (BCA) assay is a standard colorimetric method for protein concentration determination.
• Traditional BCA assays in 96-well plates show reduced performance with low sample volumes.
• A need exists for improved protein quantification methods at low volumes.

Purpose of the Study:

• To adapt the BCA assay for accurate protein quantification in reduced sample volumes.
• To investigate the use of opaque, white 384-well plates to enhance BCA assay sensitivity.
• To explore homogenous assay formats for protein concentration measurement.

Main Methods:

• Performed BCA assays in opaque, white 384-well plates using only 2 μL of sample.
• Utilized the fluorescence quenching property of the BCA chromophore (λ(max) 562 nm) by the white plates (excitation 430 nm, emission 450-600 nm).
• Investigated soluble fluorescein as an alternative to white plates for a homogenous assay format.

Main Results:

• Accurate protein concentration determination was achieved with only 2 μL of sample in 384-well plates.
• The fluorescence of the white plates was effectively quenched by the BCA chromophore, enabling sensitive detection.
• A homogenous assay format using soluble fluorescein was demonstrated as a viable alternative.

Conclusions:

• Opaque, white 384-well plates significantly improve BCA assay performance at low sample volumes.
• The fluorescence quenching principle offers a sensitive and accurate method for protein quantification.
• This adapted BCA assay method provides a valuable tool for protein analysis with minimal sample input.