Clear Native Gel Electrophoresis
Clear Native Gel Electrophoresis - Related Videos
11:38
CN-GELFrEE - Clear Native Gel-eluted Liquid Fraction Entrapment Electrophoresis
0 Views •
Cited by 12 •
2016
This protocol describes how to prepare and perform clear native gel-eluted liquid fraction entrapment electrophoresis (CN-GELFrEE), a native separation technique for non-covalent biomolecular assemblies and proteins from heterogeneous samples that is compatible with various downstream protein analysis techniques.
12:03
Blue Native Polyacrylamide Gel Electrophoresis (BN-PAGE) for Analysis of Multiprotein Complexes from Cellular Lysates
0 Views •
Cited by 92 •
2011
In this video, we describe the characterization of multiprotein complexes (MPCs) by blue native polyacrylamide gel electrophoresis (BN-PAGE). In a first dimension, dialyzed cellular lysates are separated by BN-PAGE to identify individual MPCs. In a second dimension SDS-PAGE, MPCs of interest are further subdivided to analyze their constituents by immunoblotting.
08:37
Analyzing Supercomplexes of the Mitochondrial Electron Transport Chain with Native Electrophoresis, In-gel Assays, and Electroelution
0 Views •
Cited by 15 •
2017
This protocol describes the separation of functional mitochondrial electron transport chain complexes (Cx) I-V and supercomplexes thereof using native electrophoresis to reveal information about their assembly and structure. The native gel can be subjected to immunoblotting, in-gel assays, and purification by electroelution to further characterize individual complexes.
07:31
Two-Dimensional Gel Electrophoresis
0 Views •
2023
Two-dimensional gel electrophoresis (2DGE) is a technique that can resolve thousands of biomolecules from a mixture. This technique involves two distinct separation methods that have been coupled together: isoelectric focusing (IEF) and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). This physically separates compounds across two axes of a gel by their isoelectric points (an electrochemical property) and their molecular weights. The procedure in this video covers the main...
03:10
Blue Native Polyacrylamide Gel Electrophoresis: A Non-Denaturing Separation Technique for Analysis of Intact Mitochondrial Respiratory Chain Complexes
0 Views •
2025
In this video, we demonstrate an electrophoretic technique, called blue native PAGE, for the analysis of mitochondrial respiratory protein complexes in their natively folded and functionally active state. The anionic Coomassie blue dye causes the protein complexes to become negatively charged, allowing separation based on size and structure.
FAQs
Migration reflects the combined effects of a protein complex’s net charge, size, and shape rather than a single property. Under the electric field, these characteristics determine how readily the intact assembly moves through the polyacrylamide network. Consequently, complexes with similar sizes may migrate differently if their charge or overall shape differs, making band position a composite physical measurement.
The dye can improve the electrophoretic mobility of protein complexes by contributing negative charge during migration. Its value is that it supports movement through the gel without the detergents or reducing agents associated with denaturing conditions. This balance helps researchers obtain clearer separation while retaining complex integrity for later structural or functional examination.
CN-PAGE avoids detergents and reducing agents that disrupt native protein assemblies or alter their component interactions. As a result, complexes remain available for analysis as intact systems rather than only as separated protein constituents. This distinction matters when the research question concerns oligomerization, multiprotein organization, membrane assemblies, or retained biological activity.
A band’s position in CN-PAGE reflects the combined influence of net charge, size, and shape. Therefore, migration cannot be interpreted as a simple ranking by molecular mass alone. Two assemblies with comparable mass may occupy different positions, while differences in charge or conformation can affect movement through the gel and complicate direct size-based comparisons.
The method first separates intact protein complexes in a transparent polyacrylamide gel, creating resolved assemblies for subsequent examination. Those separated complexes can then be compared through activity assays or analyzed by mass spectrometry. This sequence links electrophoretic separation with functional or compositional information, helping researchers evaluate multiprotein systems from more than one perspective.
CN-PAGE helps resolve membrane protein assemblies while maintaining the native organization needed to examine them as complexes. Because the approach avoids denaturing detergents and reducing agents, it is suited to studies focused on assembly rather than only individual subunits. Researchers can use the resulting separation to compare membrane-associated complexes before functional or compositional analyses.
By separating intact assemblies under native conditions, CN-PAGE provides a way to assess whether proteins occur as different oligomeric forms. The preserved complexes can be compared as distinct electrophoretic species, while downstream activity assays or mass spectrometry may add functional or compositional context. This makes the method useful for connecting assembly state with broader multiprotein-system behavior.
Researchers can compare the composition and functional behavior of resolved protein complexes. Electrophoresis distinguishes assemblies, mass spectrometry can support compositional analysis, and activity assays can examine retained function. Together, these readouts help relate the organization of multiprotein systems to their molecular components and biological activity without relying on electrophoretic position alone.