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5
Protein Applications
Measure Protein BCA
Working with standard curves
96
NanoDrop One User Guide
Protein standards for generating a standard curve may also be provided by the kit
manufacturer. Since the NanoDrop One pedestals 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. For example, additional standards may be required to ensure the
standard curve covers the dynamic range of the assay and the expected range of
the unknown samples.
A standard curve is required for colorimetric protein analysis.
• Each experiment requires a new standard curve.
• Prepare standards and unknown samples the same way. See the kit
manufacturer's guidelines and recommendations.
–
All reference and standards solutions should be the same buffer
used to resuspend the samples plus the same volume of reagent added to
the samples.
–
First standard is a reference measurement. The reference solution should
contain none of the analyte of interest. (The reference measurement is not
the same as a blank measurement. This application requires both.)
–
Concentration range of the standards must cover the dynamic range
of the assay and the expected range of the unknown samples. Sample
analyte concentrations are not extrapolated beyond the concentration of the
highest standard.
• Use the application setup screen to enter concentration values for the standards
and to specify how standards and samples will be measured (number of
replicates, etc.).
–
Depending on the
using two or more standards.
–
The software requires one reference measurement and allows up
to 7 standards.
–
Concentration values for standards can be entered in any order but
the standards must be measured in the order in which they were entered;
however, best practice dictates that standards be measured from the lowest
concentration of the standard analyte stock to the highest.
• For all colorimetric assays except Protein Pierce 660, blank the instrument
with DI H
O (deionized water). For Protein Pierce 660, blank with the reference
2
solution (see below).
• Measure the reference and all standards before you start analyzing
samples. (After the first sample has been measured, no additional changes are
allowed to the standard curve.)
Curve Type
setting, a standard curve can be generated
Thermo Scientific
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