Best Practices For Protein Measurements - Thermo Scientific NanoDrop One User Manual

7
Measure Protein A280

Best practices for protein measurements

56 NanoDrop One User Guide
• Isolate and purify protein samples before measurement to remove impurities. Depending
on the sample, impurities could include DNA, RNA and some buffer components. See
Preparing Samples for more information.
Note Extraction reagents that contribute absorbance between 200 nm and 280 nm
will affect measurement results if present in samples (even residual amounts).
• Ensure the sample absorbance is within the instrument's
• Choosing a blank:
– For the Protein A280, Protein A205, and Proteins & Labels applications, blank with
the same buffer solution used to resuspend the analyte of interest. The blanking
solution should be a similar pH and ionic strength as the analyte solution.
– For the Protein BCA, Protein Bradford, and Protein Lowry applications, blank with
deionized water (DI H
– For the Protein Pierce 660 application, blank with the reference solution used to
make the standard curve (reference solution should contain none of the standard
protein stock). For more information, see
• Run a blanking cycle to assess the absorbance contribution of your buffer solution. If the
buffer exhibits strong absorbance at or near the analysis wavelength (typically 280 nm or
205 nm), you may need to choose a different buffer or application, such as a colorimetric
assay (for example, BCA or Pierce 660). See
information.
Note Buffers such as Triton X, RIPA, and NDSB contribute significant absorbance
and are not compatible with direct A280 or A205 measurements.
• For micro-volume measurements:
– Ensure pedestal surfaces are properly
to pedestal surfaces.)
– Gently (but thoroughly) vortex samples before taking a measurement. Avoid
introducing bubbles when mixing and pipetting.
– Follow best practices for micro-volume measurements.
– Use a 2 μL sample volume. See
information.
• For cuvette measurements (NanoDrop One
and follow best practices for cuvette measurements.
O).
2
Working with standard
Choosing and Measuring a Blank
cleaned and
Recommended Sample Volumes
C
instruments only), use compatible cuvettes
absorbance detection limits.
curves.
for more
conditioned. (Proteins tend to stick
for more
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