User manual

Section 3- Applications
Protein Lowry
Overview
The Lowry assay is an alternative method for determining protein concentration based on the widely used and cited
Lowry procedure for protein quantitation. Like the other colorimetric assays, the Lowry Assay requires a standard
curve be generated before sample proteins can be measured.
The Lowry procedure involves reaction of protein with cupric sulfate in alkaline solution, resulting in formation of
tetradentate copper-protein complexes. The Folin-Ciocalteu Reagent is effectively reduced in proportion to the
chelated copper-complexes resulting in a water-soluble blue product that is measured at 650 nm and normalized at
405 nm. Pre-formulated reagents, utilized in the assay, are available in kit form from numerous manufacturers.
Lowry Assay Measurement Range
To accurately prepare standards, it is recommended that a 20 µL minimum sample volume and 100 µL of Lowry
reagent be used for each reaction. On the NanoDrop 2000/2000c the Lowry assay can be used to measure
samples from ~0.20 mg/mL up to 4 mg/mL.
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.
Protein standards (BSA) for generating a standard curve may also be provided by the manufacturer for the Lowry
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.
Pedestal Reconditioning
Solutions and reagents containing surfactants may “un-condition” the measurement pedestal surfaces so that the
liquid column does not form. If this occurs, use the NanoDrop Pedestal Reconditioning Compound (PR-1) as a rapid
means of reconditioning the pedestals when the surface properties have been compromised and liquid columns
break during measurement. Additional information about PR-1 may be found on our
website.
3-29