Measuring Concentration

Accurate concentration measurements are are essential when fitting equilibrium or kinetic binding constants and are important for optimal experiments characterizing protein properties.  

Protein Concentration from UV absorbance

For 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 . 

ProtParam is an EXPASY tool for calculating a number of protein parameters including UV280 extinction coefficient from a protein sequence.  It uses this formula to do so:

Molar Extinction Coefficient (M-1⋅cm-1)

ExtProt,M = NTrp*ExtTrp + NTyr*ExtTyr + NCystine*ExtCystine

Where; 
ExtTrp = 5500, ExtTyr = 1490,  ExtCystine = 125; 
NTrp, Tyr = number of Tryptophan or Tyroside residues, 
NCystine = number of cystines (disulfide bonded cysteine pairs)

The extinction coefficient in ml⋅mg-1⋅cm-1 can be calculated using the following formula: 

ExtProt = ExtProt,M / Molecular_weight

Tips for measuring concentration by absorbance:

  • Calculate the extinction coefficient for each protein.
  • Make sure each measurement is in the linear range for the UV detector being used (typically 0.1 OD - 1 OD).
  • Take replicate measurements.  I recommend taking replicate measurements at a few different dilutions for each protein.
  • 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.

 

Alternative 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 are commercially available from a variety of sources.  When measuring concentration, understand the limitations of the method being used.  

 

1  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