New polyacrylamide matrices for drift-free isoelectric focusing

Area of Science:

• Biochemistry
• Analytical Chemistry
• Polymer Science

Background:

• Isoelectric focusing (IEF) in polyacrylamide gels suffers from pH gradient decay and cathodic drift.
• This phenomenon is primarily caused by electroendo-osmotic flow (EOF) originating from fixed charges within the gel matrix.

Purpose of the Study:

• To identify the sources of fixed negative charges in polyacrylamide gels responsible for EOF.
• To investigate methods for mitigating or eliminating cathodic drift during IEF.
• To develop a stable gel matrix for improved protein separation.

Main Methods:

• Analysis of polyacrylamide gel composition to identify sources of fixed charges.
• Investigating the impact of impurities, catalyst incorporation, and hydrolysis on gel charge.
• Developing and testing a modified polyacrylamide matrix with covalently incorporated charged groups.

Main Results:

• Identified three primary sources of fixed negative charges: acrylic acid impurities, catalyst incorporation, and hydrolysis of amide groups.
• Demonstrated that conventional methods to adjust anolyte/catholyte pH are ineffective against cathodic drift.
• Developed a 'balanced' polyacrylamide gel by covalently incorporating tertiary or quaternary groups, which neutralized negative charges.

Conclusions:

• The 'balanced' polyacrylamide gel exhibited zero drift for over 5000 V.h, ensuring stable pH gradients.
• This modified gel matrix provides a robust solution for achieving equilibrium separation of protein species in isoelectric focusing.
• The findings offer a significant advancement for reproducible and high-resolution protein analysis using IEF.