User manual

Section 3- Applications
Proteins & Labels
Overview
The Proteins & Labels application can be used to determine protein concentration (A280 nm) as well as fluorescent
dye concentration (protein array conjugates). It can also be used to measure the purity of metalloproteins (such as
hemoglobin) using wavelength ratios.
Measurement Concentration Range
The NanoDrop 2000/2000c can accurately measure fluorescent-dye and protein concentrations up to 100 pmol/µl
(Cy3) and 20 mg/ml (BSA) respectively without dilution. Refer to
“Measurement Ranges” for additional information.
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.
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.
Dye/Chromophore Editor
The NanoDrop 2000/2000c software allows the user to either select from a list of pre-defined dyes or enter new
dyes using the Dye Chromophore Editor. To enter a new dye, select the Show column box (the last row). This will
activate the fields for manual entry of the appropriate information. Refer to the dye manufacturer for the appropriate
correction factors for user entered dyes. The 280 nm corrections will be automatically utilized for protein sample
concentration calculations when the respective dye is selected. Once entered, the information is saved.
To delete a user defined dye, highlight the row of interest by clicking in the grey box to the left of the (+) icon and
either use the keyboard Delete key or right-click to bring up the delete option. Pre-defined dyes, identified by the
lock icon adjacent to the dye entry in the editor, may not be edited or deleted. The default settings are Cy3 for Dye 1
and Cy5 for Dye 2.
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