Thermo Scientific NanoDrop UV-Vis spectrophotometers support protein sample quantification with applications for direct A280, A205 and colorimetric assays (see table 2 below). There are several things to consider when deciding which method to use to quantify your protein samples using a NanoDrop UV-Vis spectrophotometer.
Is your sample a purified protein? Purified protein samples can be accurately measured using direct absorbance at 280 nm. Absorbance at 280 nm is mostly due to the aromatic chains on the amino acids Tryptophan (Trp) and Tyrosine (Tyr). Protein A280 is the most popular quantification method because it is fast and simple, requires no reagents or standard curves, and consumes very little sample. Using the absorbance at 280nm (A280), protein concentration (c) is calculated using the Beer-Lambert equation A280 = c * ε * b (ε is the wavelength-dependent protein extinction coefficient, b is the pathlength). Each pure protein has a unique extinction coefficient. For accurate results, the correct protein extinction coefficient ε must be entered or the closest Sample Type must be selected. The NanoDrop One Protein Editor feature allows you to save the extinction coefficients of specific proteins so that you can customize your Sample Type options.
In addition to concentration, protein measurements on the NanoDrop One spectrophotometer deliver information about contaminants in the sample. The NanoDrop One Thermo Scientific™ Acclaro™ Sample Intelligence Technology uses mathematical algorithms to detect nucleic acids in protein samples and correct the concentration result as needed. Sample purity can also be assessed by looking at the A260/A280 value. An A260/A280 value >1 may indicate nucleic acid contamination in the protein sample.
Proteins in complex mixtures such as cell extracts or lysates are best measured using a protein colorimetric assay such as Bradford, BCA, Lowry or Thermo Scientific™ Pierce™ 660nm Assay. These assays provide protein-specific concentrations, avoiding absorbance from cell components that absorb in the UV range and would inflate A280. For buffer compatibility with each assay, check the manufacturer’s product literature. The above colorimetric assays are preconfigured applications on the NanoDrop One/OneC, NanoDrop 2000/2000c and NanoDrop 8000 instruments. (Table 2)
Does your protein/peptide contain Trp and Tyr residues? A protein’s peptide backbone absorbs light at 190-220 nm. Peptides that lack Tyr or Trp residues (and therefore cannot be measured using the A280 application) can be quantified using absorbance at 205 nm. Proteins that have significant amounts of Trp and Tyr can also be quantified using the A205 application by selecting the Scopes method option. Protein A205 is a preconfigured application for the NanoDrop One instrument but it can also be run on NanoDrop 2000 and 2000c as a custom method.
Is your protein labeled? Labeled antibodies or other fluorescently labeled proteins and metalloproteins can be quantified using the Proteins & Labels application that delivers the protein as well as the label concentration (up to 2 dyes). Proteins and Labels is available as a preconfigured application on NanoDrop One/OneC, NanoDrop 2000/2000c and NanoDrop 8000 spectrophotometers.
Table 1 below is a useful guide for using the protein quantification methods available on the NanoDrop UV-Vis spectrophotometers.Table 2 shows which protein application is available as a preconfigured method on each NanoDrop product.
The trusted Thermo Scientific NanoDrop One Microvolume UV-Vis Spectrophotometer can be used in regulated environments such as pharmaceuticals and biotechnology.
|Method||Concentration range NanoDrop One/OneC||Description*||Limitations|
*Also see Protein Assay selection guide.
|Protein Method/Feature||NanoDrop One/OneC||NanoDrop 2000/2000c||NanoDrop 8000||NanoDrop Lite|
|A205||✓||as custom method||No||No|
|Acclaro Contaminant Identification||✓||No||No||No|
|Customizable protein editor||✓||No||No||No|
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