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How do I determine protein concentration?

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  • Product Support
    • Proteins & peptides
      • Protein purification and quantitation tools

      The quickest way to estimate the amount of protein in solution is to use UV-vis to measure absorbance directly, but this is generally not very accurate or sensitive. Highly accurate quantitation of most proteins can be achieved using either a Bradford or bicinchoninic acid (BCA) assay.


      The table below summarizes the main differences between these different methods. 

      UV-vis / A280Bradford assayBCA assay
      AccuracyLow; usually used as an approximationHigh; gives accurate quantitationHigh; gives accurate quantitation
      SpeedVery fast; < 1 minFast; 20 mins1 hr, 30 mins
      Chemistry

      Tryptophan and Tyrosine residues absorb at 280 nm

      Binding of Bradford reagent to protein; complex absorbs at 595 nm

      Proteins reduce Cu2+ to Cu+; BCA chelates Cu+​; complex absorbs at 562 nm

      ConsiderationsDNA/RNA can interfere with results if present in high quantities; nucleic acids absorb at 260 nm

      Will not detect peptides without Tryptophan and Tyrosine amino acids

      SDS (>0.1 %) and other detergents should be avoided

      EDTA (>10 mM) and other strong chelating agents should be avoided

      Reducing agents should be avoided

      • If reducing agent is required, we offer reducing agent compatible formulations: ab207003 & ab207004


      UV-vis / A280

      Protein concentration can be estimated by measuring the UV absorbance at 280 nm; proteins show a strong peak here due to absorbance from Tryptophan and Tyrosine residues (commonly referred to as A280). This can readily be converted into the protein concentration using the Beer-Lambert law (see equation below). This method is used mostly for a very rough estimation of concentration, e.g. to check the success of purification.

      A = ɛcl

      • A is the absorbance (e.g. A280)
      • ɛ is the molar extinction coefficient, M-1cm-1 – this can be found from the literature
      • c is concentration in M
      • l is the path length in cm – the length of the cuvette / microplate​​​​


      Back to table



      Bradford Assay

      The Bradford assay is a colorimetric assay that relies on the binding of protein to Coomassie Brilliant Blue.

      • When the dye binds to protein in an acidic medium, a shift in absorption maximum occurs from 465 nm to 595 nm, with a change in color from brown to blue.
      • Unknown protein concentrations are estimated by reference to a standard curve generated from proteins of known concentration, typically bovine serum albumin (BSA)


      See our Bradford assay kit 

      Back to table



      BCA


      The BCA assay is a colorimetric assay that utilizes the reduction of Cu2+ ions by proteins, and subsequent binding of BCA.

      • The generated Cu+ ions form an intensely colored complex with the BCA reagent, that has a very strong absorbance at 562 nm
      • Unknown protein concentrations are estimated by reference to a standard curve generated from proteins of known concentration, typically bovine serum albumin (BSA)


      Browse our range of BCA assays

      Back to table

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