Thermo NanoDrop One - Calculations for Proteins and Labels Measurements

Measure Proteins and Labels
Thermo Scientific NanoDrop One User Guide 77
Calculations for Proteins and Labels Measurements
As with the other protein applications, Proteins & Labels
uses the Beer-Lambert equation to correlate absorbance
with concentration based on the sample’s extinction
coefficient and pathlength.
This application offers six options (shown at right) for
selecting an appropriate extinction coefficient for each
measured sample, to be used in conjunction with Beer’s
Law to calculate sample concentration.
If the extinction coefficient of the sample is known,
choose the
 + MW (molar) or 1% (mass) option and
enter the value. Otherwise, calculate the extinction
coefficient or choose the option that best matches the
sample solution.
Tip: Ideally, the extinction coefficient should be
determined empirically using a solution of the study
protein at a known concentration using the same buffer.
Available Options for Extinction Coefficient
• 1 Abs = 1 mg/mL, where sample type and/or ext. coefficient is
unknown (produces rough estimate of protein concentration)
• BSA (Bovine Serum Albumin, 6.67 L/gm-cm)
• IgG (any mammalian antibody, 13.7 L/gm-cm)
• Lysozyme (egg white lysozyme, 26.4 L/gm-cm)
• Other protein (
 + MW), user-specified molar ext. coefficient
• Other protein (
1%), user-specified mass ext. coefficient
Note: See Sample Type for details.
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.
Measured Values
A280 absorbance
Note: The absorbance value at 750 nm is subtracted from all wavelengths
in the spectrum. As a result, the absorbance at 750 nm is zero in the
displayed spectra. Also, 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
750 nm-corrected and normalized spectrum. If Analysis Correction
and Dye Correction are not selected, this is the reported A280 value
and the value used to calculate protein concentration.
•If Analysis Correction is selected, the 750-corrected, normalized and
analysis-corrected absorbance value at 280 nm is reported and used to
calculate protein concentration.
• If a Dye is used, the 750-corrected, normalized, analysis-corrected and
dye-corrected absorbance value at 280 nm is reported and used to
calculate protein concentration.