Microchip isoelectric focusing with monolithic immobilized pH gradient materials for proteins separation

Area of Science:

• Analytical Chemistry
• Biochemistry
• Materials Science

Background:

• Microchip isoelectric focusing (muCIEF) is a powerful technique for protein separation.
• Immobilized pH gradients (IPGs) offer stable and reproducible separation conditions.
• Carrier ampholytes (CAs) can interfere with downstream analyses, such as mass spectrometry.

Purpose of the Study:

• To develop monolithic immobilized pH gradient (M-IPG) materials for microchip electrophoresis.
• To evaluate the performance of M-IPG materials in microchip isoelectric focusing (muCIEF) for protein separation.
• To compare chemical and pressure mobilization for focused zone detection in muCIEF.

Main Methods:

• M-IPG materials were fabricated using photoinitiated polymerization and Ampholine attachment.
• FITC-labeled proteins (ribonuclease B, myoglobin, alpha-casein) were separated using muCIEF with M-IPG.
• Focused protein zones were detected using laser-induced fluorescence (LIF) with chemical and pressure mobilization.

Main Results:

• Proteins were well separated on M-IPG materials without the need for carrier ampholytes.
• Pressure mobilization provided superior results compared to chemical mobilization, with minimal band broadening and good peak shape.
• High detection sensitivity was achieved using LIF detection.

Conclusions:

• M-IPG materials are effective for protein enrichment and separation via muCIEF.
• The developed M-IPG-based muCIEF system eliminates interference from mobile carrier ampholytes.
• This technique is suitable for coupling with other capillary electrophoresis modes or mass spectrometry for advanced analyses.