TECHNICAL RESOURCETelephone: 800-8-PIERCE (800-874-3723) or 815-968-0747 • Fax: 815-968-7316 or 800-842-5007www.piercenet.com • Customer Service: [email protected] • Technical Assistance: [email protected] many applications involving proteins it is important either to identify fractions containing protein or to estimate theconcentration of a purified sample. Amino acids containing aromatic side chains (i.e., tyrosine, phenylalanine andtryptophan) exhibit strong UV-light absorption. Consequently, proteins and peptides absorb UV-light in proportion to theiraromatic amino acid content and total concentration. Once an absorptivity coefficient has been established for a given protein(with its fixed amino acid composition), the protein’s concentration in solution can be calculated from its absorbance.For most proteins, UV-light absorption allows detection of concentration down to 100 µg/ml. Nevertheless, estimation ofprotein concentration by UV-light absorption is not accurate for complex protein solutions (e.g., cell lysates) because thecomposition of proteins with different unknown absorption coefficients is not known. In addition, proteins are not the onlymolecules that absorb UV-light and complex solutions will usually contain compounds such as nucleic acid that will interferewith determination of protein concentration by this method. However, for aqueous protein solutions commonly used in theresearch laboratory setting, interference from other compounds is minimized by measuring absorbances at 280 nm.AbsorbanceThe ratio of radiant power transmitted (P) by a sample to the radiant power incident (P0) on the sample is called thetransmittance T:T = P/P0Absorbance (A), then, is defined as the logarithm (base 10) of the reciprocal of the transmittance:A = -log T = log (1/T)In a spectrophotometer, monochromatic plane-parallel light enters a sample at right angles to the plane-surface of the sample.In these conditions, the transmittance and absorbance of a sample depends on the molar concentration (c), light path length incentimeters (L), and molar absorptivity (ε) for the dissolved substance.1T = 10εcL or Aλ = ε c LBeer’s Law states that molar absorptivity is constant and the absorbance is proportional to concentration for a givensubstance dissolved in a given solute and measured at a given wavelength.2 Accordingly, molar absorptivities are commonlycalled molar extinction coefficients. Since transmittance and absorbance are unitless, the units for molar absorptivity mustcancel with units of measure in concentration and light path. Accordingly, molar absorptivities have units of M-1cm-1.Standard laboratory spectrophotometers are fitted for use with 1 cm width sample cuvettes; hence, the path length isgenerally assumed to be equal to one and the term is dropped altogether in most calculations.Aλ = ε c L = ε c when L = 1 cmMolar Extinction CoefficientsMolar absorptivities (= molar extinction coefficients) for many proteins are provided in the Practical Handbook ofBiochemistry and Molecular Biology.3 Expressed in this form, the extinction coefficient allows for estimation of the molarconcentration of a solution from its measured absorbance.A / ε = molar concentrationExtinction coefficients for proteins are generally reported with respect to an absorbance measured at or near a wavelength of280 nm. Although the absorption maxima for certain proteins may be at other wavelengths, 280 nm is favored becauseproteins absorb strongly there while other substances commonly in protein solutions do not.3747 N. Meridian RoadP.O. Box 117Rockford, IL 61105TR0006.0Extinction CoefficientsA guide to understanding extinction coefficients, with emphasison spectrophotometric determination of protein concentrationTelephone: 800-8-PIERCE (800-874-3723) or 815-968-0747 • Fax: 815-968-7316 or 800-842-5007www.piercenet.com • Customer Service: [email protected] • Technical Assistance: [email protected] for 1% SolutionsMany sources, including the reference cited above, provide absorbance (A280nm) values for 1% (= 1 g/100 ml) solutionsmeasured in a 1 cm path length. These values can be understood as percent solution extinction coefficients (εpercent) havingunits of (g/100 ml)-1 cm-1 instead of M-1cm-1. Consequently, when these values are applied as extinction coefficients in thegeneral formula, the units for concentration, c, are percent solution (i.e., 1% = 1 g/100 ml = 10 mg/ml).A / εpercent = percent concentrationIf one wishes to report concentration in terms of mg/ml, then an adjustment factor of 10 must be made when using thesepercent solution extinction coefficients (i.e., one must convert from 10 mg/ml units to 1 mg/ml concentration units).(A / εpercent) 10 = concentration in mg/mlThe relationship between Molar Extinction Coefficient (εmolar) and Percent Extinction Coefficient (εpercent) is as follows(εmolar) 10 = (εpercent) × (molecular weight of protein)ExamplesA. Proteins and Protein Mixtures with Unknown Extinction CoefficientsIf no extinction coefficient information exists for a protein or protein mixture of interest, and a rough estimate of proteinconcentration is required for a solution that has no other interfering substances, assume εpercent = 10. Most protein extinctioncoefficients (εpercent) fall in the range 4.0-24.0.3 Therefore, although any given protein can vary significantly from εpercent = 10,the average for a mixture of many different proteins will likely be close to 10.B. ImmunoglobulinsMost mammalian antibody types (i.e., immunoglobulins) have protein extinction coefficients (εpercent) in the range 13-15.Therefore, rough estimation of antibody concentration should be made assuming εpercent = 14.C. Bovine Serum Albumin (BSA)Pierce Albumin Standard Ampules (Bovine Albumin Fraction V, Product No. 23209) are provided as a 2 mg/ml solution in0.9% NaCl. The product is calibrated by absorbance at 280 nm to a BSA Fraction V standard from the National Institute ofStandards and Technology (NIST) having a reported percent solution absorbance (= εpercent) equal to 6.67.Therefore, the predicted absorbance at 280 nm for the Pierce Albumin Standard, assuming exactly 2 mg/ml and the statedεpercent, isεpercent c L / 10 = A[(6.67)(2.000)(1)] / 10 = 1.334Suppose that relative to a water reference a researcher obtains a 280 nm absorbance reading of 1.346 for the AlbuminStandard. The calculated concentration is(A
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