Thermo NanoDrop One - Page 62

Measure Protein A280
56 NanoDrop One User Guide Thermo Scientific
Best practices for protein measurements
• 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.
• Ensure the sample absorbance is within the instrument’s absorbance detection limits.
• 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
2
O).
– 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 Working with standard curves.
•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 Choosing and Measuring a Blank for more
information.
• For micro-volume measurements:
– Ensure pedestal surfaces are properly cleaned and conditioned. (Proteins tend to stick
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 Recommended Sample Volumes for more
information.
• For cuvette measurements (NanoDrop One
C
instruments only), use compatible cuvettes
and follow best practices for cuvette measurements.
Note Extraction reagents that contribute absorbance between 200 nm and 280 nm
will affect measurement results if present in samples (even residual amounts).
Note Buffers such as Triton X, RIPA, and NDSB contribute significant absorbance
and are not compatible with direct A280 or A205 measurements.