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Labeling DNA Probes03:31

Labeling DNA Probes

DNA probes are fragments of DNA labeled with a reporter tag to enable their detection or purification. The resulting labeled DNA probes can then hybridize to target nucleic acid sequences through complementary base-pairing, and may be used to recover or identify these regions.
Radioisotopes, fluorophores, or small molecule binding partners like biotin or digoxigenin, are the most widely used reporter tags for labeling DNA probes. These labels can be attached to the probe DNA molecule via...

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Selective Labelling of Cell-surface Proteins using CyDye DIGE Fluor Minimal Dyes
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2D DIGE saturation labeling for minute sample amounts.

Georg J Arnold1, Thomas Fröhlich

  • 1Laboratory for Functional Genome Analysis LAFUGA, Gene Center, Ludwig-Maximilians-University, Munich, Germany. arnold@genzentrum.uni-muenchen.de

Methods in Molecular Biology (Clifton, N.J.)
|February 8, 2012
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Summary

Two-dimensional saturation difference gel electrophoresis (2D DIGE) offers ultrasensitive protein quantification for minimal sample amounts. This method enhances reproducibility and sensitivity in proteomic analysis, crucial for complex biological samples.

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Area of Science:

  • Proteomics
  • Biochemistry
  • Analytical Chemistry

Background:

  • Two-dimensional difference gel electrophoresis (2D DIGE) improves protein quantification reproducibility, sensitivity, and dynamic range.
  • Saturation DIGE utilizes cysteine labeling with cyanine dyes for enhanced protein detection and quantification in 2D gel electrophoresis.

Purpose of the Study:

  • To provide guidelines for establishing ultrasensitive 2D saturation DIGE in proteomic workgroups.
  • To detail experimental design, labeling chemistry, sample preparation, and artifact avoidance.
  • To present a step-by-step protocol for sample preparation, image analysis, and statistical evaluation.

Main Methods:

  • Utilizing 2D DIGE with saturation labeling of cysteine sulfhydryl groups.
  • Analyzing complex mammalian cell or tissue samples with minimal protein amounts (micrograms).
  • Generating preparative saturation DIGE gels for mass spectrometry-based identification.

Main Results:

  • Achieving high sensitivity for protein detection and quantification using minimal sample inputs.
  • Enabling the analysis of approximately 2,000 analytes per 2D gel.
  • Demonstrating 2D saturation DIGE as a preferred method for quantitative proteome analysis with limited samples.

Conclusions:

  • 2D saturation DIGE is a powerful, ultrasensitive technique for quantitative proteome analysis when sample amounts are limited.
  • Successful implementation requires strict adherence to reaction conditions and careful handling of low-input samples.
  • The described protocol supports the adoption of this technically demanding yet highly sensitive proteomic method.