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Related Experiment Video

Updated: Jun 21, 2026

Quantification of Proteins Using Peptide Immunoaffinity Enrichment Coupled with Mass Spectrometry
06:09

Quantification of Proteins Using Peptide Immunoaffinity Enrichment Coupled with Mass Spectrometry

Published on: July 31, 2011

Protein quantification in complex mixtures by solid phase single-molecule counting.

Lee A Tessler1, Jeffrey G Reifenberger, Robi D Mitra

  • 1Center for Genome Sciences, Department of Genetics, Washington University in St. Louis School of Medicine, 4444 Forest Park Avenue, St. Louis, Missouri 63108, USA.

Analytical Chemistry
|July 16, 2009
PubMed
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This summary is machine-generated.

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This study introduces a new method for counting single protein molecules on surfaces by detecting bound antibodies. This technique offers sensitive and accurate quantification of protein abundance in complex samples like serum.

Area of Science:

  • Biochemistry
  • Analytical Chemistry
  • Surface Science

Background:

  • Accurate quantification of single protein molecules on surfaces is crucial for various biological and diagnostic applications.
  • Previous methods faced challenges with robust single-molecule detection due to nonspecific adsorption and limited antibody binding times.

Purpose of the Study:

  • To develop and validate a reliable procedure for quantifying single surface-immobilized protein molecules.
  • To overcome limitations in current single-molecule detection techniques for surface-bound proteins.

Main Methods:

  • Utilizing a chemically adsorbed bovine serum albumin (BSA) surface to minimize nonspecific protein adsorption.
  • Employing fluorescent antibodies for direct counting of bound antibodies to quantify target proteins.

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Published on: November 15, 2017

Proteome-wide Quantification of Labeling Homogeneity at the Single Molecule Level
08:29

Proteome-wide Quantification of Labeling Homogeneity at the Single Molecule Level

Published on: April 19, 2019

Related Experiment Videos

Last Updated: Jun 21, 2026

Quantification of Proteins Using Peptide Immunoaffinity Enrichment Coupled with Mass Spectrometry
06:09

Quantification of Proteins Using Peptide Immunoaffinity Enrichment Coupled with Mass Spectrometry

Published on: July 31, 2011

Deep Proteome Profiling by Isobaric Labeling, Extensive Liquid Chromatography, Mass Spectrometry, and Software-assisted Quantification
10:37

Deep Proteome Profiling by Isobaric Labeling, Extensive Liquid Chromatography, Mass Spectrometry, and Software-assisted Quantification

Published on: November 15, 2017

Proteome-wide Quantification of Labeling Homogeneity at the Single Molecule Level
08:29

Proteome-wide Quantification of Labeling Homogeneity at the Single Molecule Level

Published on: April 19, 2019

  • Systematic investigation of parameters affecting single-molecule detection sensitivity and robustness.

    • Main Results:

    • The BSA surface enabled efficient detection of single protein molecules with fluorescent antibodies.
    • Antibodies demonstrated sufficient binding duration for imaging billions of molecules.
    • Accurate quantification of protein abundance was achieved by counting bound antibodies, showing a linear relationship (R(2) = 0.98) with immobilized protein molecules.
    • High precision (1-5% CV) allowed for reliable detection of small abundance changes (7%).

    Conclusions:

    • The developed procedure provides high sensitivity and accurate quantification of single, surface-immobilized protein molecules.
    • This method is robust and effective even in real-world serum samples.
    • The potential for multiplexed single-molecule immunoassays was demonstrated through multiple probing of flow cells.