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SDS-PAGE

SDS-PAGE

Gel electrophoresis is a method that separates biological macromolecules like nucleic acids or proteins by forcing them to pass through a gel matrix under an electric field.
A variation of gel electrophoresis, termed polyacrylamide gel electrophoresis (PAGE), is commonly used for separating proteins according to their molecular size by passing them through a polyacrylamide gel. Because of the varying charges associated with amino acid side chains, PAGE can be used to separate intact...

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Related Experiment Video

Updated: Feb 25, 2026

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

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Published on: November 15, 2017

Acid/Salt/pH Gradient Improved Resolution and Sensitivity in Proteomics Study Using 2D SCX-RP LC-MS.

Ming-Zhi Zhu^1,2, Na Li¹, Yi-Tong Wang¹

¹State Key Laboratory for Quality Research of Chinese Medicines, Macau University of Science and Technology , Macao, China.

Journal of Proteome Research

|July 29, 2017

Summary

This study introduces an improved 2D SCX-RP LC method using acid/salt/pH gradients for enhanced peptide and protein identification in proteomics. The new approach significantly boosts resolution and reduces fraction overlap compared to traditional salt elution.

Keywords:

SCX-RP LC acid/salt/pH gradient overlap resolution sensitivity

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Published on: December 4, 2017

Area of Science:

Proteomics
Chromatography
Analytical Chemistry

Background:

Strong cation exchange (SCX) chromatography in proteomics suffers from poor resolution and nonspecific hydrophobic interactions.
High salt concentrations in SCX limit its application in online 2D SCX-Reverse Phase Liquid Chromatography (RP LC).

Purpose of the Study:

To enhance peptide and protein identification in proteomics by improving SCX chromatography resolution and reducing hydrophobic interactions.
To develop an optimized online 2D SCX-RP LC method utilizing novel elution strategies.

Main Methods:

Exploited online 2D SCX-RP LC by combining acid, salt, and pH gradients for peptide elution.
Incorporated 50% acetonitrile (ACN) in the elution buffer to eliminate hydrophobic interactions.
Reduced volatile salt concentration to 50 mM.

Main Results:

Acid/salt/pH gradient elution demonstrated superior resolution, sensitivity, and uniform fraction distribution.
Achieved a 62.5% increase in protein and a 60.6% increase in unique peptide identification compared to salt elution alone.
Significantly minimized fraction overlap and improved identification of acidic and hydrophilic peptides.

Conclusions:

The novel acid/salt/pH gradient elution method significantly advances SCX chromatography for proteomics.
This approach overcomes limitations of traditional SCX, leading to more comprehensive and accurate peptide and protein identification.
The method offers a more sensitive and robust platform for proteomic analyses.