Quantify your proteins with NanoDrop instruments

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?
Is your sample a purified protein?

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)

FIG 1. Protein BCA data on the NanoDrop One 
Does your protein/peptide contain Trp and Tyr residues?
Does your protein/peptide contain Trp and Tyr residues?

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?
SI-labeled-protein-80x97

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.

FIG 2. Fluorescein – labeled IgG measured with Proteins and Labels application on the NanoDrop One

Can NanoDrop One be used in regulated environments?

The trusted Thermo Scientific NanoDrop One Microvolume UV-Vis Spectrophotometer can be used in regulated environments such as pharmaceuticals and biotechnology.

Learn about NanoDrop One Compliance solution.

Table 1: NanoDrop One protein methods

MethodConcentration range NanoDrop One/OneCDescription*Limitations
A280
  • pedestal: 0.06-820 mg/mL BSA (0.03-400 mg/mL IgG)
  • cuvette: 0.006-2.38 mg/mL BSA
  • Direct measurement, quick and easy
  • Best method for pure proteins that contain Trp and Tyr residues.
  • Uses Beer's Law to calculate concentration. For accurate results the correct extinction coefficient must be entered
  • Does not require standard curve
  • When measuring protein mixtures use 1 Abs=1 mg/mL “sample type”.
  • Contaminants or buffers that absorb ~280 nm will affect protein concentration calculation.
  • Thermo Scientific™ Acclaro™Sample Intelligence Technology detects nucleic acid contaminants in protein samples and corrects concentration results.
A205
  • varies from peptide to peptide. Approximate range 0.003 - 10.74 mg/mL 
  • Measures peptide backbone absorbance at 205 nm. For peptides that lack or have few Trp and Tyr residues
  • Scopes method option can be used to measure proteins that have significant amounts of Trp and Tyr.
  • More sensitive than A280 since A205, molar absorptivity is high.
  • High salt protein buffers such as PBS and TE absorb in the low UV. Use a low salt buffer like the Brij® buffer diluted to 0.01%.
Pierece 660
  • 50-2000 μg/mL BSA (15:1 reagent:sample vol)
  • 25-1000 μg/mL BSA (7.5:1 reagent:sample vol)
  • The most accurate, fast and easy colorimetric assay. 
  • Compatible with most detergents, reducing agents and Laemmli loading buffer. 
  • Assay done at room temperature with short incubation times.
  • Reagents stored at room temperature. 
  • Requires standard curve. Expanded linear range for standard curve compared to Bradford.
  • Less protein-to-protein variation than the Bradford assay
Bradford
  • 100-8000 μg/mL BSA (50:1 reagent:sample vol)
  • 15-100 μg/mL BSA (1:1 reagent:sample vol)
  • Measures Coomassie Blue shift at 595 nm when bound to proteins. 
  • Fast and easy assay done at room temperature.
  • Linear range 100-1000 μg/mL BSA ( for 50:1 ratio ).
  • Requires standard curve.
  • Surfactants may cause reagent precipitation. 
  • Coomassie-dye binding assay exhibits twice as much protein-to-protein variability as the BCA assay
BCA
  • 200-8000 μg/mL BSA (20:1 reagent:sample vol) 
  • 10-200 μg/mL BSA (1:1 reagent:sample vol)
  • Measures Cu-BCA chelates at 562 nm formed in the presence of proteins. 
  • Often used for more dilute samples.
  • Compatible with most surfactants at concentrations up to 5%.
  • Less protein-to-protein variation compared to Coomassie methods.
  • Requires standard curve.
  • Reducing agents, copper chelators and high capacity buffers can interfere with assay.
Lowry
  • 200-4000 μg/mL BSA
  • Measures Cupric sulfate-tartrate complexes at 650 nm.
  • Modified Lowry at 750 nm can be run as a custom method
  • Complexes can be measured from 650-750 nm.
  • Requires standard curve.
  • Potassium ions and detergents precipitate reagent.
  • Chelating agents, reducing agents and free thiols interfere with this assay.

*Also see Protein Assay selection guide.

Table 2: Protein measurements by NanoDrop instrument

Protein Method/FeatureNanoDrop One/OneCNanoDrop 2000/2000cNanoDrop 8000NanoDrop Lite
A280✓✓✓✓
A205✓as custom methodNoNo
Pierce 660✓✓✓No
Bradford✓✓✓No
BCA✓✓✓No
Lowry✓✓✓No
A260/A280✓✓✓No
Acclaro Contaminant Identification✓NoNoNo
Customizable protein editor✓NoNoNo

For research use only. Not for use in diagnostic procedures. Brij is a registered trademark of UNIQEMA AMERICAS LLC. All other trademarks are the property of Thermo Fisher Scientific Inc., and its subsidiaries. 

Resources

Support