Thermo Scientific NanoDrop One, User Manual

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7
Measure Protein A280
66 NanoDrop One User Guide Thermo Scientific
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.
Most sources report extinction coefficients for proteins
measured at or near 280 nm in phosphate or other
physiologic buffer. These values provide sufficient
accuracy for routine assessments of protein
concentration. Published Extinction Coefficients
Published extinction coefficients for proteins may be reported as:
• wavelength-dependent molar absorptivity (or extinction) coefficient
(

) with units of M
-1
cm
-1
• percent solution extinction coefficient (

1%) with units of
(g/100 mL)
-1
cm
-1
(i.e., 1% or 1 g/100 mL solution measured in a
1 cm cuvette)
• protein absorbance values for 0.1% (i.e., 1 mg/mL) solutions
Tip : Assess published values carefully to ensure unit of measure is applied
correctly.
The equation at the right shows the relationship
between molar extinction coefficient (

molar
) and
percent extinction coefficient (

1%).
Conversions Between

molar
and

1%
(

molar
) * 10 = (

1%) * (MW
protein
)
Example: To determine percent solution extinction coefficient (

1%) for a
protein that has a molar extinction coefficient of 43,824 M
-1
cm
-1
and a
molecular weight (MW) of 66,400 daltons (Da), rearrange and solve the
above equation as follows:

1% = (

molar
* 10) / (MW
protein
)

1% = (43,824 * 10) / 66,400 Da)

1% = 6.6 g/100 mL