Fluorescence Spectroscopy

Claudia Y. Lee1

1 SDIX, LLC, Newark, Delaware
Publication Name:  Current Protocols Essential Laboratory Techniques
Unit Number:  Unit 2.4
DOI:  10.1002/9780470089941.et0204s10
Online Posting Date:  May, 2015
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Abstract

Fluorescence is an extremely powerful tool in modern biology, physics, and chemistry laboratories. This unit begins with the physics of fluorescence, the biological applications of fluorescence, and the mechanisms behind spectrometers and fluorometers, followed by strategies to choose an appropriate fluorophore according to the instrumentation at hand. The first part of the unit walks the reader through the acquisition of an absorption spectrum and explains how it can be used to measure the degree of labeling on conjugated proteins. The discussion then extends into how to obtain fluorescence emission spectra, which can be utilized as fluorescence indicators for protein detection. © 2015 by John Wiley & Sons, Inc.

Keywords: fluorescence; immunolabeling; absorption spectrum; emission spectrum; fluorometer; spectrometer

     
 
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Table of Contents

  • Introduction
  • Strategic Planning
  • Safety Considerations
  • Basic Protocol 1: Labeling Proteins with Fluorescently Tagged NHS‐Esters
  • Support Protocol 1: Measuring the Extinction Coefficient of Protein
  • Support Protocol 2: Preparing the Reactive Dye Stock Solution
  • Support Protocol 3: Purification of Samples Greater than 100 μg by Gravity Filtration
  • Support Protocol 4: Purification of Samples less than 100 μg by Spin‐Column Filtration
  • Alternate Protocol 1: Detecting Protein Using ANS
  • Support Protocol 5: Preparing ANS Solution
  • Reagents and Solutions
  • Understanding Results
  • Troubleshooting
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Labeling Proteins with Fluorescently Tagged NHS‐Esters

  Materials
  • 1 mg/ml protein sample in phosphate‐buffered saline (PBS; see recipe)
  • 1 M sodium bicarbonate (see recipe)
  • Reactive dye stock solution (see protocol 3)
  • Size exclusion (desalting) gravity flow column
  • Phosphate buffered saline (PBS), pH 7.2 (see recipe)
  • Spectrophotometer (also see unit 2.1; Morris, )
  • Cuvette
  • Aluminum foil

Support Protocol 1: Measuring the Extinction Coefficient of Protein

  Materials
  • Reactive dye in powder form (e.g., Alexa Fluor 488 carboxylic acid, succinimidyl ester [Invitrogen]; FITC [Thermo Fisher Scientific]; rhodamine‐maleimide [Biotium])
  • DMSO, anhydrous grade
  • Disposable cuvettes
  • Spectrophotometer (also see unit 2.1; Morris, )

Support Protocol 2: Preparing the Reactive Dye Stock Solution

  Materials
  • Labeled protein solution ( protocol 3)
  • Phosphate‐buffered saline (PBS; see recipe)
  • Chromatography column [unit 6.1 (Mallik et al., ) and unit 6.2 (Chakravarti et al., 2008b), e.g., PD‐10 desalting column (GE Healthcare Life Sciences)
  • Long‐wavelength (365‐nm) handheld UV lamp

Support Protocol 3: Purification of Samples Greater than 100 μg by Gravity Filtration

  Materials
  • Labeled protein solution ( protocol 3)
  • Size‐exclusion gel (Bio‐Gel P‐6, BioRad)
  • Spin columns: low‐protein‐binding centrifuge filter column (VWR Scientific, cat. no. 82031‐356)
  • Microcentrifuge, preferably with swinging‐bucket rotor
  • Spectrophotometer (also see unit 2.1; Morris, )

Support Protocol 4: Purification of Samples less than 100 μg by Spin‐Column Filtration

  Materials
  • 10 μM ANS in deionized H 2O ( protocol 7)
  • Sample that might contain protein
  • Spectrofluorometer

Alternate Protocol 1: Detecting Protein Using ANS

  Materials
  • 1,8‐ anilinonaphthalene‐8‐sulfonic acid (ANS; Sigma, cat. no. A‐1028)
  • Spectrophotometer
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Figures

Videos

Literature Cited

Literature Cited
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Key Reference
  Lakowicz, J.R. 1999. Principles of Fluorescence Spectroscopy, 2nd ed. Kluwer Academic/Plenum Publishers, New York.
  The most complete textbook on fluorescence, covering the physics, history, and experiments.
Internet Resources
  http://www.lifetechnologies.com/us/en/home/references/molecular‐probes‐the‐handbook.html
  The Handbook from Life Technologies, containing spectra and protocols for fluorescent probes.
  http://www.iss.com/resources/reference/data_tables/LifetimeDataFluorophores.html
  A list of lifetime values for commonly used fluorophores.
  http://www.lifetechnologies.com/us/en/home/life‐science/cell‐analysis/labeling‐chemistry/fluorescence‐spectraviewer.html
  A useful tool for visualizing the spectra of fluorophores, excitation light sources, and detection.
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