Isoelectric Focusing
Isoelectric Focusing - Related Videos
03:11
Liquid Phase Isoelectric Focusing: An Electrophoresis Technique to Separate Bioactive Molecules from Crude Gymnema sylvestre Extract
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2025
This video describes a technique to separate complex molecules from plant extracts using liquid phase isoelectric focusing. This technique helps purify complex biomolecules in their native state which can be used for various downstream applications.
07:58
Highly Sensitive and Quantitative Detection of Proteins and Their Isoforms by Capillary Isoelectric Focusing Method
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2018
Capillary isoelectric focusing is an antibody-based, ultrasensitive, high throughput technique, enabling detailed characterization of proteins and their isoforms from extremely small biological samples. The following describes a protocol for detection and quantification of specific proteins and their isoforms in an automated and robotized manner.
09:57
Separation of Bioactive Small Molecules, Peptides from Natural Sources and Proteins from Microbes by Preparative Isoelectric Focusing (IEF) Method
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Cited by 4 •
2020
The objective is to fractionate and isolate bioactive small molecules, peptides from a complex plant extract, and proteins from pathogenic microbes by employing liquid-phase isoelectric focusing (IEF) method. Further, the separated molecules were identified and their bioactivity confirmed.
10:19
Induction of an Isoelectric Brain State to Investigate the Impact of Endogenous Synaptic Activity on Neuronal Excitability In Vivo
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Cited by 3 •
2016
This procedure performs long-lasting in vivo intracellular recordings from single neurons during physiologically relevant cerebral states and after complete abolition of ongoing electrical activities, resulting in an isoelectric brain state. The physiological constants of the animal are carefully monitored during the transition to the artificial comatose condition.
09:30
Focused Ion Beams
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2023
Source: Sina Shahbazmohamadi and Peiman Shahbeigi-Roodposhti-Roodposhti, School of Engineering, University of Connecticut, Storrs, CT As electron microscopes become more complex and widely used in research labs, it becomes more of a necessity to introduce their capabilities. Focused ion beam (FIB) is an instrument that can be employed in order to fabricate, trim, analyze and characterize materials on mico- and nano-scales in a wide variety of fields from nano-electronics to medicine. FIB...
FAQs
The stabilized gradient provides a spatial map of pH across the separation medium. As molecules move through this map, their charge changes according to the local pH, allowing each one to locate the region corresponding to its isoelectric point. This converts differences in charge behavior into distinct physical positions that can be observed as bands.
Amphoteric molecules can carry different electrical charges as the surrounding pH changes. That property allows an applied electric field to move them through the gradient while they carry a net charge. Their changing charge is essential because it directs migration toward the pH region where the molecule reaches its isoelectric point.
Charge variants can be distinguished because a change in a protein’s charge behavior changes the pH at which it has no net charge. Under the applied field, variants therefore focus at different positions in the gradient. Separate bands provide evidence of charge heterogeneity, supporting characterization of variant forms within a biological sample.
A basic workflow establishes a stabilized pH gradient, applies an electric field, and allows the sample’s amphoteric molecules to migrate through that gradient. Molecules accumulate at their matching isoelectric locations, producing focused bands. Researchers can then interpret band positions to estimate isoelectric points and evaluate charge-related differences within the sample.
Researchers can use the band pattern to characterize protein charge variants, estimate their isoelectric points, and assess sample complexity. These outcomes provide a charge-based view of biological samples, distinct from the molecular-mass separation used in the second dimension of two-dimensional gel electrophoresis for proteomic analysis.
In two-dimensional gel electrophoresis, isoelectric focusing provides the first separation dimension. Proteins are initially resolved according to isoelectric point, then separated by molecular mass in the subsequent dimension. Combining these two properties produces a more informative proteomic analysis than relying on charge-related position or molecular mass alone.