Quantitation of Nucleic Acids and Proteins

Sean R. Gallagher1, Philippe Desjardins2

1 UVP, LLC, Upland, California, 2 Thermo Scientific NanoDrop Products, Wilmington, Delaware
Publication Name:  Current Protocols Essential Laboratory Techniques
Unit Number:  Unit 2.2
DOI:  10.1002/9780470089941.et0202s5
Online Posting Date:  July, 2011
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Abstract

Reliable quantitation of nanogram and microgram amounts of DNA and RNA in solution is essential to researchers in molecular biology. Two methods for direct absorbance measurements at 260 nm are described—the first is a traditional cuvette‐based method, and the second is a microvolume method that requires no cuvettes or capillaries. In addition, three fluorescence techniques using Hoechst 33258, ethidium bromide, and PicoGreen reagent are presented in this unit. These five procedures cover a range from 5 to 10 ng/ml DNA to 15,000 µg/ml DNA. Reliable quantitation of proteins is possible using several types of assays. UV spectroscopy is the simplest approach but is limited in sensitivity. More sensitive assays that use Coomassie blue binding, bicinchoninic acid (BCA), and the Lowry reaction are also described. All assays are prone to amino acid composition errors and interference from assay solution components. Flow charts and tables to help with appropriate method selection are included. Curr. Protoc. Essential Lab. Tech. 5:2.2.1‐2.2.36. © 2011 by John Wiley & Sons, Inc.

Keywords: nucleic acid; protein; quantitation; fluorescence; colorimetric; electrophoresis; standards

     
 
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Table of Contents

  • Overview and Principles
  • Strategic Planning
  • Protocols: Nucleic Acid Quantification
  • Basic Protocol 1: Traditional Detection of Nucleic Acids Using Absorption Spectroscopy
  • Basic Protocol 2: Microvolume Detection of Nucleic Acids Using Absorption Spectroscopy
  • Alternate Protocol 1: DNA Detection Using the DNA‐Binding Fluorochrome Hoechst 33258
  • Alternate Protocol 2: DNA and RNA Detection with Ethidium Bromide Fluorescence
  • Alternate Protocol 3: DNA Detection Using PicoGreen dsDNA Quantitation Reagent
  • Protocols: Protein Quantification
  • Basic Protocol 3: Lowry Protein Assay
  • Alternate Protocol 4: Lowry Protein Assay, Reduced Volume
  • Support Protocol 1: Deoxycholate‐Trichloroacetic Acid (DOC‐TCA) Sample Precipitation for Removal of Interfering Compounds and Sample Concentration
  • Basic Protocol 4: BCA Protein Assay
  • Basic Protocol 5: Coomassie Blue Protein Assay (Bradford Assay)
  • Basic Protocol 6: Traditional UV Spectrophotometry
  • Alternate Protocol 5: Protein Quantitation with UV Spectroscopy and Correction for Like‐Acid Contamination
  • Basic Protocol 7: Microvolume UV Spectrophotometry
  • Protocol Common to Nucleic Acids and Proteins
  • Basic Protocol 8: Gel‐Based Quantitation of Protein and Nucleic Acids
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Traditional Detection of Nucleic Acids Using Absorption Spectroscopy

  Materials
  • Deionized water or appropriate buffer as sample diluent and blank
  • Sample containing nucleic acids
  • NanoDrop 2000 or 2000 c Spectrophotometer (Thermo Scientific NanoDrop Products, http://www.thermoscientific.com/nanodrop)
  • Nuclease‐free, low‐retention pipet tips

Basic Protocol 2: Microvolume Detection of Nucleic Acids Using Absorption Spectroscopy

  • Hoechst 33258 assay solution (working solution; see recipe)
  • DNA Standards (e.g., lambda, calf thymus; Bio‐Rad, Invitrogen or DNAQF Kit, Sigma Aldrich)
  • Dedicated filter fluorometer (e.g., DQ 300, Hoefer; QuantiFluor, Promega) or scanning fluorescence spectrophotometer (RF‐5301 PC, Shimadzu; F‐2500, Hitachi; FluoroMax, Horiba)
  • Fluorometric square glass cuvettes or disposable acrylic cuvettes (Sarstedt)
  • Teflon stir rod

Alternate Protocol 1: DNA Detection Using the DNA‐Binding Fluorochrome Hoechst 33258

  • Ethidium bromide assay solution (see recipe)
CAUTION: Please follow established safety procedures for handling DNA binding dyes such ethidium bromide ( appendix 1A).

Alternate Protocol 2: DNA and RNA Detection with Ethidium Bromide Fluorescence

  • PicoGreen dsDNA quantitation kit (Invitrogen) containing:
    • PicoGreen dsDNA quantitation reagent (Component A), 1 ml solution in DMSO (store up to 6 months at –20°C, protected from light)
    • 20× TE (component B), 25 ml of 200 mM Tris⋅Cl/20 mM EDTA, pH 7.5: store up to 6 months at 4°C (preferred) or up to 6 months at –20°C
    • Lambda DNA standard (component C), 1 ml of 100 µg/ml in TE: store up to 6 months at 4°C (preferred) or up to 6 months at –20°C
  • DNase‐free water (e.g., Millipore), sterile
  • Spectrofluorometer or fluorescence microplate reader

Alternate Protocol 3: DNA Detection Using PicoGreen dsDNA Quantitation Reagent

  Materials
  • 0.5 mg/ml standard protein stock solution (see recipe)
  • Sample protein
  • Copper‐tartrate‐carbonate (CTC; see recipe)
  • Lowry reagents A and B (see recipe)
  • 5‐ml test tubes
  • Spectrophotometer with visible light source

Basic Protocol 3: Lowry Protein Assay

  Materials
  • See protocol 6

Alternate Protocol 4: Lowry Protein Assay, Reduced Volume

  Materials
  • Protein sample (dilute or with interfering compounds)
  • 0.15% (w/v) deoxycholate (DOC)
  • 72% (w/v) trichloroacetic acid (TCA)
  • 1.5‐ml microcentrifuge tubes
  • Filter paper

Support Protocol 1: Deoxycholate‐Trichloroacetic Acid (DOC‐TCA) Sample Precipitation for Removal of Interfering Compounds and Sample Concentration

  Materials
  • 0.5 mg/ml standard protein stock solution (see recipe)
  • Protein sample
  • Bicinchoninic acid (BCA) standard working reagent (SWR; see recipe or commercially available, e.g., Pierce)
  • 5‐ml test tubes
  • 37°C or 60°C water bath
  • Spectrophotometer with visible light source

Basic Protocol 4: BCA Protein Assay

  Materials
  • Sample protein
  • 0.5 mg/ml standard protein stock solution (see recipe)
  • 1 M sodium hydroxide
  • Coomassie protein reagent (see recipe or available commercially, e.g., Pierce)
  • Disposable plastic cuvettes or test tubes
  • Spectrophotometer with visible light source

Basic Protocol 5: Coomassie Blue Protein Assay (Bradford Assay)

  Materials
  • Sample protein dissolved in compatible buffer (see Table 2.2.3)
  • 0 to 3 mg/ml standard protein stock solution in compatible buffer (see Table 2.2.3)
  • Protein standard
  • Spectrophotometer with UV light source
  • UV transparent quartz or methacrylate cuvettes

Basic Protocol 6: Traditional UV Spectrophotometry

  Materials
  • Protein sample
  • Deionized water or appropriate buffer for sample diluent and blank
  • NanoDrop 2000 or 2000 c spectrophotometer (Thermo Fisher Scientific)

Alternate Protocol 5: Protein Quantitation with UV Spectroscopy and Correction for Like‐Acid Contamination

  Materials
  • Stained gel containing protein or nucleic acid samples and quantitated protein or nucleic acid standards
  • Protein electrophoresis standards (e.g., Pierce, Sigma Aldrich, or Fermentas); prequantitated protein standards for solution assays are acceptable
  • Nucleic acids electrophoresis standards (e.g., NEB, Sigma Aldrich, or Fermentas)
  • CCD imaging system with image analysis software (unit 7.5)
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Figures

Videos

Literature Cited

   Applied Biosystems. 1987. User Bulletin Issue 11, Model No. 370. Applied Biosystems, Foster City, Calif.
   Bradford, M.M. 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein‐dye binding. Anal. Biochem. 72:248‐254.
   Cesarone, C.F., Bolognesi, C., and Santi, L. 1979. Improved microfluorometric DNA determination in biological material using 33258 Hoechst. Anal. Biochem. 100:188‐197.
   Daxhelet, G.A., Coene, M.M., Hoet, P.P., and Cocito, C.G. 1989. Spectrofluorometry of dyes with DNAs of different base composition and conformation. Anal. Biochem. 179:401‐403.
   Desjardins, P.R. and Conklin, D.S. 2011. Microvolume quantitation of nucleic acids. Curr. Protoc. Mol. Biol. 93:A.3J.1‐A.3J.16.
   Desjardins, P., Hansen, J.B., and Allen, M. 2009. Microvolume spectrophotometric and fluorometric determination of protein concentration. Curr. Protoc. Protein Sci. 55:3.10.1‐3.10.16.
   Dunn, B. 1995. Quantitative amino acid analysis. Curr. Protoc. Protein Sci. 0:3.2.1‐3.2.3.
   Gallagher, S.R. and Desjardins, P.R. 2006. Quantitation of DNA and RNA with absorption and fluorescence spectroscopy. Curr. Protoc. Mol. Biol. 76:A.3D.1‐A.3D.21.
   Grimsley, G.R. and Pace, C.N.f 2003. Spectrophotometric determination of protein concentration. Curr. Protoc. Protein Sci. 33:3.1.1‐3.1.9.
   Groves, W.E., Davis, F.C. Jr., and Sells, B.H. 1968. Spectrophotometric determination of microgram quantities of protein without nucleic acid interference. Anal. Biochem. 22:195‐210.
   Jones, L.J., Haugland, R.P., and Singer, V.L. 2003. Development and characterization of the NanoOrange protein quantitation assay: A fluorescence‐based assay of proteins in solution. BioTechniques 34:850‐858.
   Labarca, C. and Paigen, K. 1980. A simple, rapid, and sensitive DNA assay procedure. Anal. Biochem. 102:344‐352.
   Layne, E. 1957. Spectrophotometric and turbidimetric methods for measuring proteins. Methods Enzymol. 3:447‐454.
   Le Pecq, J.‐B. 1971. Use of ethidium bromide for separation and determination of nucleic acids of various conformational forms and measurement of their associated enzymes. In Methods of Biochemical Analysis, Vol. 20 (D. Glick, ed.) pp. 41‐86. John Wiley & Sons, New York.
   Lowry, O.H., Rosebrough, N.J., Farr, A.L., and Randall, R.J. 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193:265‐275.
   Marmur, J. and Doty, P. 1962. Determination of the base composition of deoxyribonucleic acid from its thermal denaturation temperature. J. Molec. Biol. 5:109‐118.
   Neidle, S. 2001. DNA minor‐groove recognition by small molecules. Nat. Prod. Rep. 18:291‐309.
   Olson, B.J.S.C. and Markwell, J. 2007. Assays for determination of protein concentration. Curr. Protoc. Protein Sci. 48:3.4.1‐3.4.29.
   Peterson, G.L. 1977. A simplification of the protein assay method of Lowry et al. which is more generally applicable. Anal. Biochem. 83:346‐356.
   Peterson, G.L. 1979. Review of the Folin phenol protein quantitation method of Lowry, Rosebrough, Farr and Randall. Anal. Biochem. 100:201‐220.
   Peterson, G.L. 1983. Determination of total protein. Methods Enzymol. 91:95‐119.
   Portugal, J. and Waring, M.J. 1988. Assignment of DNA binding sites for 4′,6‐diamidine‐2‐phenylindole and bisbenzimide (Hoechst 33258): A comparative footprinting study. Biochem. Biophys. Acta 949:158‐168.
   Sapan, C.V., Lundblad, R.L., and Price, N.C. 1999. Colorimetric protein assay techniques. Biotechnol. Appl. Biochem. 29:99‐108.
   Scopes, R.K. 1974. Measurement of protein by spectrophotometry at 205 nm. Anal. Biochem. 59:277‐282.
   Smith, P.K., Krohn, R.I., Hermanson, G.T., Mallia, A.K., Gartner, F.H., Provenzano, M.D., Fujimoto, E.K., Goeke, N.M., Olson, B.J., and Klenk, D.C. 1985. Measurement of protein using bicinchoninic acid. Anal. Biochem. 150:76‐85.
   Stoscheck, C.M. 1990. Quantitation of protein. Methods Enzymol. 182:50‐68.
   Stout, D.L. and Becker, F.F. 1982. Fluorometric quantitation of single‐stranded DNA: A method applicable to the technique of alkaline elution. Anal. Biochem. 127:302‐307.
   Struhl, K. 1993. Reagents and radioisotopes used to manipulate nucleic acids. Curr. Protoc. Mol. Biol. 9:3.4.1‐3.4.11.
   Van Lancker, M. and Gheyssens, L.C. 1986. A comparison of four frequently used assays for quantitative determination of DNA. Anal. Lett. 19:615‐623.
   Voet, D., Gratzer, W.B., Cox, R.A., and Doty, P. 1963. Absorption spectra of nucleotides, polynucleotides, and nucleic acids in the far ultraviolet. Biopolymers 1:193‐208.
   Wallace, R.B. and Miyada, C.G. 1987. Oligonucleotide probes for the screening of recombinant DNA libraries. In Methods of Enzymology, Vol. 152: Guide to Molecular Cloning Techniques (S.L. Berger and A.R. Kimmel, eds.) pp. 432‐442. Academic Press, San Diego.
   Warburg, O. and Christian, W. 1941. Isolierung and Kristallisation des Gärungsferments Enolase. Biochem. Z. 310:310‐384.
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Key References
  Labarca and Paigen, 1980. See above.
  Contains a detailed description of the Hoechst 33258 fluorometric DNA assay.
  Peterson, 1997. See above.
  Describes in detail the Lowry assay used in this chapter.
  Sapan et al., 1999. See above.
  An excellent overview of the used and limits of protein assays.
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