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Related Experiment Videos

High-content proteomics: fluorescence multiplexing using an integrated, high-sensitivity, multiwavelength

Mary F Lopez1, Alvydas Mikulskis, Eva Golenko

  • 1PerkinElmer Life and Analytical Sciences, 549 Albany Street, Boston, MA 02118, USA. mary.lopez@perkinelmer.com

Proteomics
|July 23, 2003
PubMed
Summary

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Fluorescent protein detection offers superior sensitivity and quantitation for proteomics, replacing older methods. Advanced imaging systems enable multiplexing for deeper proteomic data analysis from single samples.

Area of Science:

  • Proteomics
  • Biotechnology
  • Analytical Chemistry

Background:

  • Two-dimensional gel electrophoresis (2-D PAGE) coupled with mass spectrometry (MS) is a standard for protein identification.
  • Fluorescent detection methods are increasingly preferred over colorimetric and radiometric techniques for gel and blot analysis.
  • Fluorescence offers enhanced sensitivity, quantitation, dynamic range, speed, safety, and protein-to-protein consistency.

Purpose of the Study:

  • To highlight the advantages of fluorescent detection in proteomics.
  • To introduce advanced instrumentation for sensitive, high-resolution fluorescent imaging.
  • To demonstrate the utility of multiplexing strategies for comprehensive proteomic analysis.

Main Methods:

  • Utilized a charge-coupled device (CCD) based imager (ProXPRESS) for multilabel fluorescent imaging.

Related Experiment Videos

  • Configured the imager for a wide range of excitation and emission wavelengths (380-700 nm and UV).
  • Employed xenon lamp or UV excitation sources for image acquisition.

    • Main Results:

    • Demonstrated the capability for precise, sensitive, high-resolution image acquisition.
    • Showcased multiplexing ability to simultaneously detect total protein, enzyme activities, and protein modifications using different fluorescent probes.
    • Highlighted the compatibility of fluorescent detection with subsequent MS identification.

    Conclusions:

    • Fluorescent detection provides significant advantages for protein analysis in proteomics.
    • Advanced imaging instrumentation like the ProXPRESS facilitates efficient multicolor fluorescence detection and multiplexing.
    • Multiplexing strategies are crucial for in-depth proteomic studies, enabling richer data extraction from single samples.