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Subcellular Fractionation

Subcellular Fractionation

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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Related Experiment Video

Updated: May 4, 2026

Detection of Protein Ubiquitination Sites by Peptide Enrichment and Mass Spectrometry

Detection of Protein Ubiquitination Sites by Peptide Enrichment and Mass Spectrometry

Published on: March 23, 2020

Methods for Intracellular Peptidomic Analysis.

Rosangela A S Eichler¹, Luiz Felipe Martucci¹, Leandro M de Castro²

¹Department of Pharmacology, Biomedical Sciences Institute, University of São Paulo, São Paulo, SP, Brazil.

Methods in Molecular Biology (Clifton, N.J.)

|March 29, 2024

Summary

This summary is machine-generated.

Extracting intracellular peptides requires rapid inactivation of degrading enzymes. Heating samples to 80°C works for most tissues, but plasma needs alternative methods to prevent clotting and preserve peptide integrity.

Keywords:

Brain Formaldehyde Intracellular peptides Plasma Stable isotope

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Last Updated: May 4, 2026

Detection of Protein Ubiquitination Sites by Peptide Enrichment and Mass Spectrometry

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Sample Preparation and Relative Quantitation using Reductive Methylation of Amines for Peptidomics Studies

Sample Preparation and Relative Quantitation using Reductive Methylation of Amines for Peptidomics Studies

Published on: November 4, 2021

Area of Science:

Biochemistry
Molecular Biology
Proteomics

Background:

Intracellular peptides are bioactive molecules generated from protein degradation.
Efficient extraction of these peptides is crucial for their study.
Proteases and peptidases in biological samples can rapidly degrade peptides.

Purpose of the Study:

To describe effective methods for extracting intracellular peptides.
To address challenges in inactivating enzymes that degrade peptides during extraction.

Main Methods:

Rapid inactivation of proteases and peptidases is essential.
Heating biological sources (tissue, cells, fluids) at 80°C for 20 minutes effectively denatures enzymes.
Avoiding acidification at high temperatures prevents nonspecific hydrolysis, particularly of Asp-Pro bonds.

Main Results:

Heat inactivation at 80°C is sufficient for most biological samples.
Plasma samples require alternative enzyme inactivation methods due to heat-induced clotting above 55°C.
Specific protocols for successful intracellular peptide extraction are detailed.

Conclusions:

Effective intracellular peptide extraction relies on prompt enzyme inactivation.
Sample type dictates the appropriate method for enzyme inactivation.
The described methods facilitate the recovery of bioactive intracellular peptides.