User manual

Section 3- Applications
Protein BCA
Overview
The BCA (Bicinchoninic Acid) assay is a colorimetric method for determining the total protein concentration in
unpurified protein samples. It is often used for dilute protein solutions and/or proteins in the presence of
components that have significant UV (280 nm) absorbance. Unlike the Protein A280 application, the Protein BCA
application requires a standard curve be generated before sample protein concentrations can be measured.
The method uses bicinchoninic acid (BCA) as the detection reagent for Cu+1, which is formed when Cu+2 is
reduced by protein in an alkaline environment. A purple-colored reaction product is formed by the chelation of two
molecules of BCA with one cuprous ion (Cu+1). The resulting Cu-BCA chelate formed in the presence of protein is
measured at 562 nm and normalized at 750 nm. Pre-formulated reagents of BCA and CuSO
4
, are available in kit
form from Thermo Fisher Scientific.
BCA Assay Measurement Ranges
Commercial BCA Protein kit manufacturers typically outline procedures for different protein concentration ranges:
• A regular assay uses a 20:1 reagent / sample volume ratio to detect a concentration range between ~0.20
mg/mL to 8.0 mg/mL (BSA). When setting up an assay for pedestal measurements, a minimum sample
volume of 4 µL in 80 µL of BCA reagent is recommended.
• A mini assay uses a 1:1 reagent / sample volume ratio to detect a concentration range between 0.01 mg/mL
to 0.20 mg/mL (BSA). To prepare sufficient volume of these 1:1 mixtures for pedestal measurements, a
minimum sample volume of 10 µL and 10 µL of BCA reagent (in a PCR tube) is recommended.
Follow the assay kit manufacturer’s recommendations for all standards and samples (unknowns). Ensure each is
subjected to the same timing and temperature throughout the assay.
Note: If running the assay at 60°C, doubling the volumes may afford greater insurance against skewed results
from evaporation / condensation within the sealed reaction tube.
Protein standards (BSA) for generating a standard curve may also be provided by the manufacturer for the BCA
assay. Since the NanoDrop 2000/2000c pedestal option can measure higher protein concentrations than traditional
cuvette-based spectrophotometers, you may need to supply your own protein standards at higher concentrations
than provided by the manufacturer.
Sample Volume Requirements
Although sample size is not critical, it is essential that a liquid column is formed when using the pedestal option so
that the gap between the upper and lower measurement pedestals is bridged with sample.
The dominant factor determining the surface tension of a droplet is the hydrogen bonding of the lattice of water
molecules in solution. Generally, all additives including protein, buffer salts and detergent-like molecules may
reduce the surface tension by interfering with the hydrogen bonding between water molecules. Although 1 µL
volumes are usually sufficient for most sample measurements, increasing the sample size to 2 µL for protein
measurements will ensure proper column formation for samples with reduced surface tension.
When using the cuvette option, it is essential that sufficient sample volume be used to ensure that the light path is
passing through a representative portion of the sample. The optical beam (2 mm) is directed 8.5 mm above the
bottom of the cuvette. Refer to the cuvette manufacturer for recommended volumes.
3-24