Thermo NanoDrop One - Page 73

Measure Protein A280
Thermo Scientific NanoDrop One User Guide 67
To determine concentration (c) of a sample in mg/mL, use
the equation at the right and a conversion factor of 10.
Tip: The NanoDrop One software includes the
conversion factor when reporting protein concentrations.
Conversions Between g/100 mL and mg/mL
C
protein
in mg/mL = (A / 1%) * 10
Example: If measured absorbance for a protein sample at 280 nm relative
to the reference is 5.8 A, protein concentration can be calculated as:
C
protein
= (A / 1%) * 10
C
protein
= (5.8/6.6 g/100 mL) * 10
C
protein
= 8.79 mg/mL
Calculated protein concentrations are based on the
absorbance value at 280 nm, the selected (or entered)
extinction coefficient and the sample pathlength. A
single-point baseline correction (or analysis correction)
may be applied.
Concentration is reported in mass units. Calculators are
available on the Internet to convert concentration from
mass to molar units based on sample sequence.
Absorbance values at 260 nm and 280 nm are used to
calculate purity ratios for the measured protein samples.
Purity ratios are sensitive to the presence of
contaminants in the sample, such as residual solvents
and reagents typically used during sample purification.
Measured Values
A280 absorbance
Note: For micro-volume absorbance measurements and measurements
taken with nonstandard (other than 10 mm) cuvettes, the spectra are
normalized to a 10 mm pathlength equivalent.
• Protein absorbance values are measured at 280 nm using the
normalized spectrum. If Baseline Correction is not selected, this is the
reported A280 value and the value used to calculate protein
concentration.
•If Baseline Correction is selected, the normalized and
baseline-corrected absorbance value at 280 nm is reported and used to
calculate protein concentration.
A260 absorbance
• Normalized and baseline-corrected (if selected) absorbance value at
260 nm is also reported.
Sample Pathlength
• For micro-volume measurements, the software selects the optimal
pathlength (between 1.0 mm and 0.03 mm) based on sample
absorbance at the analysis wavelength.
• For cuvette measurements, pathlength is determined by the cuvette
Pathlength setting in the software (see General Settings).
• Displayed spectra and absorbance values are normalized to a 10 mm
pathlength equivalent.
•