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Related Experiment Video

Updated: Dec 22, 2025

Density Gradient Ultracentrifugation for Investigating Endocytic Recycling in Mammalian Cells

Density Gradient Ultracentrifugation for Investigating Endocytic Recycling in Mammalian Cells

Published on: June 30, 2021

Density Gradient Ultracentrifugation to Isolate Endogenous Protein Complexes after Affinity Capture.

Javier Fernandez-Martinez¹, John LaCava¹, Michael P Rout¹

¹Laboratory of Cellular and Structural Biology, The Rockefeller University, New York, New York 10065.

Cold Spring Harbor Protocols

|July 3, 2016

Summary

This protocol details isolating native protein complexes using density gradient ultracentrifugation. This method effectively separates intact complexes from contaminants, yielding a pure sample for further analysis.

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Last Updated: Dec 22, 2025

Density Gradient Ultracentrifugation for Investigating Endocytic Recycling in Mammalian Cells

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Protein Complex Affinity Capture from Cryomilled Mammalian Cells

Protein Complex Affinity Capture from Cryomilled Mammalian Cells

Published on: December 9, 2016

Area of Science:

Biochemistry
Molecular Biology
Proteomics

Background:

Affinity capture and native elution often yield mixed protein complex samples.
Purification is necessary to isolate homogeneous complexes from degradation fragments and elution reagents.

Purpose of the Study:

To describe a protocol for isolating native protein complexes.
To detail the use of density gradient ultracentrifugation for purification.

Main Methods:

Utilizing rate-zonal density gradient ultracentrifugation.
Generating density gradients using inert solutes like sucrose or glycerol.
Performing ultracentrifugation in solvents compatible with affinity capture and elution.

Main Results:

Separation of protein complexes based on hydrodynamic volume.
Isolation of homogeneous protein complexes from heterogeneous mixtures.
Preservation of protein complex structure and activity during purification.

Conclusions:

Density gradient ultracentrifugation is a powerful technique for purifying native protein complexes.
This method enables the isolation of intact complexes, free from degradation products and reagents.
The protocol ensures the integrity and functionality of the purified protein assemblies.