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Calculations for Protein A205 Measurements
As with the other protein applications,
Proteins A205 uses the
equation
to correlate absorbance with
concentration based on the sample's
extinction coefficient and pathlength.
This application offers three 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
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.
Calculated protein concentrations are
based on the absorbance value at 205 nm,
the selected (or entered) extinction
coefficient and the sample pathlength. A
single-point baseline correction may also
be applied.
Concentration is reported in mass units.
Calculators are available on the Internet to
convert concentration from mass to molar
units based on the sample sequence.
Thermo Scientific
Beer-Lambert
Available Options for Extinction Coefficient
• 31, assumes
0.1% (1 mg/mL) at 205 nm = 31
• Scopes, assumes
120 * (A280/A205)
• Other protein, enter mass extinction coefficient in
L/gm-cm for 1 mg/mL (
Note: See
Sample Type
Measured Values
A205 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 205 nm
using the normalized spectrum. If Baseline Correction is
not selected, this is the reported A205 value and the
value used to calculate protein concentration.
• If
Baseline Correction
baseline-corrected absorbance value at 205 nm is
reported and used to calculate protein concentration.
A280 absorbance
• Normalized and baseline-corrected (if selected)
absorbance value at 280 nm is also reported.
5
Protein Applications
Measure Protein A205
0.1% (1 mg/mL) at 205 nm = 27 +
0.1%) protein solution
for details.
is selected, the normalized and
NanoDrop One User Guide
83
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