Measuring Concentration
Accurate concentration measurements are are essential when fitting equilibrium or kinetic binding constants and are important for optimal experiments characterizing protein properties.
Preferred Method for measuring protein concentration: Protein Absorbance
For purified proteins with aromatic residues, UV absorbance at 280 is one of the more reliable methods for measuring concentration. To measure protein concentration from UV, an extinction coefficient is needed and can be calulated from the sum of amino acid absorbances.
Other Methods for measuring protein concentration
Copper chelation methods (BCA assay) and Protein-dye methods using absorbance detection (eg. Bradford assay using coomassie) or fluorescence detection kits are commercially available from a variety of sources. When measuring concentration, understand the limitations of the method being used.
| Method | Features | Example Assays |
|---|---|---|
UV Absorbance Direct measure of protein absorbance using calculated extinction coefficient A = ε c l | •Most accurate method for known pure proteins •Requires aromatic residues (Trp, Tyr) •No protein standard required | OD 280 nm Molar Extinction Coefficient (M-1⋅cm-1) calculated from W, Y, C-C |
Copper-Chelation Protein-copper chelation and secondary detection of reduced copper (Biuret reaction) | •Less protein–protein variation than colorimetric •Incompatible with reagents that reduce copper, reducing agents •Compatible with surfactants | BCA Assays OD 562 nm Lowry Assays OD 660 nm |
Colorimetric Protein-dye binding and direct detection of the color change | •Fast and easy •Higher protein–protein variation •Incompatible with surfactants | Bradford Assay (Coomassie) OD 595 nm |
Fluorescent Protein-dye binding and direct detection of fluorescence associated with the bound dye | •Excellent sensitivity •Requires specialized equipment •Timing is not a critical factor | ThermoFisher: Qubit Protein Assay NanoOrange Protein Assay |
adapted from thermofisher Protein-assay-technical-handook.pdf
For purified proteins with aromatic residues, UV absorbance at 280 is one of the more reliable methods for measuring concentration. To measure protein concentration from UV, an extinction coefficient is needed and can be calulated from the sum of amino acid absorbances at 280 nm.1 For proteins without Trp or Tyr, absorbance at 205 nm may be used with care.2
Tips for measuring concentration by absorbance:
- Calculate the extinction coefficient for each protein.
- Use UV280 (Pace et al.) when your protein has tryptophan or tyrosine. For protein or peptides without Trp or Tyr, use UV205 (Anthis and Clore).
- Make sure each measurement is in the linear range for the UV detector being used (typically 0.1 OD - 1 OD for UV280).
- Take replicate measurements. It's even better to take replicate measurements at a few different dilutions for each protein and average those in the linear range for your detector.
- Make sure to use a well-match buffer blank. This is critical when working with buffers that may absorb at 280, such as DTT-containing buffers or when measuring absorbance at 205, where many buffers absorb.
UV280 Molar Extinction Coefficient (M-1⋅cm-1) 1
ProtParam is an EXPASY tool for calculating a number of protein parameters including UV280 extinction coefficient from a protein sequence. It uses this formula from Pace et al:
ε280M = NTrp*εTrp + NTyr*εTyr + NCystine*εCystine
ε280M = NTrp(5500) + NTyr(1490) + NCystine(125)
where:
- εTrp = 5500, εTyr = 1490, εCystine = 125;
- NTrp, Tyr = number of Tryptophan or Tyroside residues,
- NCystine = number of cystines (disulfide bonded cysteine pairs)
Extinction coefficient in ml⋅mg-1⋅cm-1 can be calculated using the following formula:
- ExtProt = ExtProt,M / Molecular_weight
UV205 Molar Extinction Coefficient (M-1⋅cm-1) 2
ε205M = Npeptide(2780) + NTrp(20400) + NPhe(8600) + NTyr(6080) + NHis(5200) + NMet(1830) + NArg(1350) + NCystine(690) + NGln(400) + NAsn(400)
where:
- εpeptide = 2780, εTrp = 20400, εPhe = 8600, εTyr = 6080, εHis = 5200, εMet = 1830, εArg = 1350, εCys = 690, εGln = 400, εAsn = 400
- Npeptide = number of peptide bonds
- Naa = number aa residues
British Peptides Labs - Molarity Calculator includes a tool for calculating both ε280 and ε205.
- How to measure and predict the molar absorption coefficient of a protein. Pace CN, Vajdos F, Fee L, Grimsley G, Gray T. Protein Sci. 1995 Nov;4(11):2411-23. PMID: 8563639
- Sequence-specific determination of protein and peptide concentrations by absorbance at 205 nm. Anthis NJ, Clore GM. Protein Sci. 2013 Jun;22(6):851-8. PMID: 23526461