User manual

Section 3- Applications
Protein Bradford
Overview
The Bradford Assay is a commonly used method for determining protein concentration. It is often used for more
dilute protein solutions where lower detection sensitivity is needed and/or in the presence of components that also
have significant UV (280 nm) absorbance. Like the other colorimetric assays, the Bradford assay requires a
standard curve be generated before sample proteins can be measured.
The Bradford procedure uses the protein-induced absorbance shift of Coomassie Blue dye to 595 nm as a measure
of protein concentration. The bound protein-dye complex is measured at 595 nm and normalized at 750 nm. A
single stabilized reagent mixture containing Coomassie Blue dye, alcohol, and surfactant in kit form is available
from numerous manufacturers.
Bradford Assay Measurement Ranges
Commercial Bradford protein kit manufacturers typically outline procedures for different protein concentration
ranges:
• A regular assay uses a 50:1 reagent / sample volume ratio to detect a concentration range between ~0.1
mg/mL to 8.0 mg/mL (BSA). The best linearity is in the 0.01- 1 mg/mL range. When setting up an assay for
pedestal measurements, a minimum sample volume of 4 µL in 200 µL of Bradford reagent is
recommended.
• A mini assay uses a 1:1 reagent / sample volume ratio to detect a concentration range between 15 µg/mL –
125 µg/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), ensuring they are
subjected to the same timing and temperature throughout the assay.
Protein standards (BSA) for generating a standard curve may also be provided by the manufacturer for the Bradford
assay.
Note: 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.
Coomassie dye-dye and Coomassie dye-protein aggregates are frequently encountered in Coomassie dye-based
protein assays. Particulates often form with increasing development time, which can cause significant fluctuations in
Absorbance readings. It is also important to note the total analyte (protein-dye) signal at 595 nm is limited to ~ 0.150
A as a result of the 1.0 mm pathlength of the pedestal, the Bradford (Coomassie dye) reagent concentration, and
the acidic pH. Making measurements in triplicate of standards and samples (unknowns) is good practice,
particularly with the limited assay signal obtained with the Bradford Assay. Using the cuvette option of the
NanoDrop 2000 c will result in a larger signal.
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.
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