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Related Concept Videos

Centrifugation01:05

Centrifugation

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Centrifugation is a separation technique based on differences in density or size. It is commonly used to separate solids from aqueous interferents. During centrifugation, the sample is placed in centrifugation tubes and spun at high angular velocity, which allows centrifugal force to act differentially on the different densities or masses of the components. After spinning, the supernatant liquid is decanted. Depending on the specific application, either the pellet or the supernatant is retained...
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Subcellular Fractionation01:32

Subcellular Fractionation

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The homogenate obtained after cell lysis contains various membrane-bound organelles that can be further separated into pure fractions by subcellular fractionation. These isolates are used to study specific cellular components, analyze localized protein activity, and are even employed in diagnostics. Fractionation is typically achieved using centrifugation methods, the most common being density-gradient and differential centrifugation.
Differential Centrifugation
Differential centrifugation is...
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SDS-PAGE01:27

SDS-PAGE

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Gel electrophoresis is a method that separates biological macromolecules like nucleic acids or proteins by forcing them to pass through a gel matrix under an electric field.
A variation of gel electrophoresis, termed  polyacrylamide gel electrophoresis (PAGE), is commonly used for separating proteins according to their molecular size by passing them through a polyacrylamide gel. Because of the varying charges associated with amino acid side chains, PAGE can be used to separate intact...
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Related Experiment Video

Updated: Mar 7, 2026

Polysome Profiling without Gradient Makers or Fractionation Systems
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Zonal Sedimentation Analysis on Sucrose Gradients.

Javier G Magadán1

  • 1Laboratory of Viral Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD, USA.

Bio-Protocol
|February 28, 2017
PubMed
Summary

Zonal sedimentation analysis determines the molecular size of proteins and protein complexes. This protocol details evaluating the oligomeric state of influenza A virus hemagglutinin (HA) trimers.

Area of Science:

  • Biochemistry
  • Structural Biology
  • Virology

Background:

  • Zonal sedimentation analysis is a biophysical technique used to estimate the molecular size of proteins and protein complexes.
  • This method is effective under nondenaturing conditions, preserving native protein structures.
  • Influenza A virus (IAV) hemagglutinin (HA) is a key surface glycoprotein, often studied as a trimer.

Purpose of the Study:

  • To provide a detailed, step-by-step protocol for evaluating the oligomeric state of proteins.
  • To specifically demonstrate the application of zonal sedimentation analysis for characterizing recombinant HA trimers.

Main Methods:

  • Sucrose gradient ultracentrifugation under nondenaturing conditions.
  • Constant speed centrifugation to separate molecules based on size and shape.

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  • Analysis of fractions to determine the sedimentation coefficient and infer molecular size.

    • Main Results:

    • The protocol enables the estimation of molecular size for individual proteins and protein complexes.
    • Successfully applied to assess the oligomeric state of recombinant influenza A virus hemagglutinin (HA) trimers.
    • Demonstrates the suitability of zonal sedimentation for globular proteins.

    Conclusions:

    • Zonal sedimentation analysis is a robust method for determining protein oligomeric states.
    • The described protocol offers a reliable approach for structural characterization of viral glycoproteins like HA.
    • This technique is valuable for understanding the structural dynamics of protein complexes.