Microvolume Spectrophotometric and Fluorometric Determination of Protein Concentration

Philippe Desjardins1, Joel B. Hansen1, Michael Allen1

1 Thermo Fisher Scientific/NanoDrop Products, Wilmington, Delaware
Publication Name:  Current Protocols in Protein Science
Unit Number:  Unit 3.10
DOI:  10.1002/0471140864.ps0310s55
Online Posting Date:  February, 2009
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Abstract

Methods for determining protein concentration that use progressively smaller amounts of material are continually being developed. A new way of minimizing the amount of sample used for spectroscopic analysis is providing more opportunities for greater quality control. Traditional spectrophotometric and fluorometric methods for determination of protein concentrations have long required placing samples into containment devices such as cuvettes or capillaries. A microsample retention system is changing that paradigm by using natural surface tension properties to capture and hold microvolume samples in place during measurement without traditional containment devices. The advantage of such a system is to dramatically reduce the amount of sample required (1 to 2 µl) while greatly increasing the dynamic range of protein concentrations that can be measured. Modifications to classic protein concentration determination protocols are presented to provide a microvolume alternative to traditional cuvette‐based methods. Curr. Protoc. Protein Sci. 55:3.10.1‐3.10.16. © 2009 by John Wiley & Sons, Inc.

Keywords: microvolume; microvolume spectrophotometer; microvolume fluorospectrometer; microsample; microvolume spectroscopy; protein concentration; microsample analysis; microvolume analysis

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Microvolume Spectrophotometric Determination of Protein Concentration Using A280 Absorbance
  • Microvolume Spectrophotometric Determination of Protein Concentration Using Colorimetric Assays
  • Basic Protocol 2: BCA Protein Assay Using a Microvolume Spectrophotometer
  • Microvolume Fluorometric Determination of Protein Concentration
  • Basic Protocol 3: Fluorometric Protein Assay Using a Microvolume Fluorospectrometer
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Microvolume Spectrophotometric Determination of Protein Concentration Using A280 Absorbance

  Materials
  • Deionized water
  • Protein sample
  • NanoDrop 1000 spectrophotometer (Thermo Fisher Scientific)
  • Laboratory wipes

Basic Protocol 2: BCA Protein Assay Using a Microvolume Spectrophotometer

  Materials
  • BCA protein assay kit, reducing agent compatible (Pierce cat. no. 23250) containing:
    • BCA reagent A
    • BCA reagent B
    • Compatibility reagent
    • Reconstitution buffer
    • Albumin standard
  • Ultrapure water
  • Protein samples
  • 1.5‐ml microcentrifuge tubes
  • 37°C heating block or water bath
  • NanoDrop 1000 spectrophotometer (Thermo Fisher Scientific)
  • Laboratory wipes
NOTE: Commercial BCA protein kit manufacturers typically outline procedures for two different protein concentration ranges. The regular assay uses a 20:1 reagent/sample volume ratio. To accurately prepare standards for the regular assay, use a minimum sample volume of 4 µl in 80 µl of BCA reagent (larger sample volume is preferable). The mini assay uses a 1:1 reagent/sample volume ratio. To prepare sufficient volume of these 1:1 mixtures, use a minimum of 10 µl of sample and 10 µl of BCA reagent in a PCR tube. Using the same pipettor for both volumes will eliminate any pipet‐to‐pipet differences.NOTE: If the assay is to be performed at 60°C, doubling the volumes may provide greater insurance against obtaining skewed results arising from evaporation or condensation within the sealed reaction tube.NOTE: Some buffer components and reagents as well as detergents may cause the pedestal surfaces to become “unconditioned.” For example, routine use of the Bradford reagent may result in difficulty forming columns with 1‐µl samples. Although the instrument was designed for 1‐µl samples, using larger volumes (1.5 to 2.0 µl) will often overcome the inherent surface tension properties associated with some detergent‐based samples, allowing for proper column formation. Use the NanoDrop pedestal reconditioning compound (PR‐1) as a rapid means of reconditioning the pedestals when the surface properties have been compromised and liquid columns break during measurement.

Basic Protocol 3: Fluorometric Protein Assay Using a Microvolume Fluorospectrometer

  Materials
  • Quant‐iT protein assay kit (Molecular Probes cat. no. Q33210)containing:
    • Quant‐iT protein reagent
    • BSA standards (0, 25, 50, 100, 200, 300, 400, and 500 ng/µl)
    • Quant‐iT protein buffer
  • Nuclease‐free, deionized water
  • Nuclease‐free amber or foil‐wrapped 1.5‐ml polypropylene microcentrifuge tubes
  • Microvolume fluorospectrometer (NanoDrop ND‐3300 fluorospectrometer)
  • 2‐µl pipettor (low‐retention, nuclease‐free tips)
  • Laboratory wipes (e.g., Kimwipes)
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Figures

Videos

Literature Cited

Literature Cited
   Aitken, A. and Learmonth, M. 1996. Protein determination by UV absorption. In Protein Protocols Handbook (J.M. Walker, ed). Springer, Secaucus, N.J.
   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.
   Lowry, O.H., Rosebrough, N.J., Farr, A., and Randall, R.J. 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193:265‐275.
   Ohnishi, S.T. and Barr, J.K. 1978. A simplified method of quantitating protein using the biuret and phenol reagents. Anal. Biochem. 86:193‐200.
   Reisner, A.H., Nemes, P., and Bucholtz, C. 1975. Use of Coomassie Brilliant Blue G250 perchloric acid solution for staining in electrophoresis and isoelectric focusing on polyacrylamide gels. Anal. Biochem. 64:509‐516.
   Simonian, M.H. and Smith, J.A. 2006. Spectrophotometric and colorimetric determination of protein concentration. Curr. Protoc. Mol. Biol. 10:1A.17.
   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. Meth. Enzymol. 182:50‐68.
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