ArticleJournal of visualized experiments : JoVE2017
Nitrogen Cavitation and Differential Centrifugation Allows for Monitoring the Distribution of Peripheral Membrane Proteins in Cultured Cells.
Article in Journal of visualized experiments : JoVE, 2017. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 14 papers.
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Who cites it
14 citing papers in PubMed.
- Nitrogen cavitation enables rapid and high-yield preparation of functional cell-membrane-derived vesicles.Nanoscale advances · 2026Article
- Delivery of TCM Monomers Using Blood Cell Membrane Coated Biomimetic Nanoparticles to Target CVD.International journal of nanomedicine · 2026Review
- Blocking C-terminal processing of KRAS4b via a direct covalent attack on the CaaX-box cysteine.Proceedings of the National Academy of Sciences of the United States of America · 2025Article
- Optimization of Cell Membrane Purification for the Preparation and Characterization of Cell Membrane Liposomes.Small methods · 2024Article
- AP-1B regulates interactions of epithelial cells and intraepithelial lymphocytes in the intestine.Cellular and molecular life sciences : CMLS · 2024Article
- An Extracellular/Membrane-Bound S100P Pool Regulates Motility and Invasion of Human Extravillous Trophoblast Lines and Primary Cells.Biomolecules · 2023Article
- Cell-Free Expression System Derived from a Near-Minimal Synthetic Bacterium.ACS synthetic biology · 2023Article
- RAB27B controls palmitoylation-dependent NRAS trafficking and signaling in myeloid leukemia.The Journal of clinical investigation · 2023Article
- Article
- NRAS is unique among RAS proteins in requiring ICMT for trafficking to the plasma membrane.Life science alliance · 2021Article
- Scaffold association factor B (SAFB) is required for expression of prenyltransferases and RAS membrane association.Proceedings of the National Academy of Sciences of the United States of America · 2020Article
- KRAS4A directly regulates hexokinase 1.Nature · 2019Article
- Isolation of Neutrophil Nuclei for Use in NETosis Assays.Bio-protocol · 2019Article
- Demarcating the membrane damage for the extraction of functional mitochondria.Microsystems & nanoengineering · 2018Article
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Authors and funding
2 authors.
Funding
Abstract
Cultured cells are useful for studying the subcellular distribution of proteins, including peripheral membrane proteins. Genetically encoded fluorescently tagged proteins have revolutionized the study of subcellular protein distribution. However, it is difficult to quantify the distribution with fluorescent microscopy, especially when proteins are partially cytosolic. Moreover, it is often important to study endogenous proteins. Biochemical assays such as immunoblots remain the gold standard for quantification of protein distribution after subcellular fractionation. Although there are commercial kits that aim to isolate cytosolic or certain membrane fractions, most of these kits are based on extraction with detergents, which may be unsuitable for studying peripheral membrane proteins that are easily extracted from membranes. Here we present a detergent-free protocol for cellular homogenization by nitrogen cavitation and subsequent separation of cytosolic and membrane-bound proteins by ultracentrifugation. We confirm the separation of subcellular organelles in soluble and pellet fractions across different cell types, and compare protein extraction among several common non-detergent-based mechanical homogenization methods. Among several advantages of nitrogen cavitation is the superior efficiency of cellular disruption with minimal physical and chemical damage to delicate organelles. Combined with ultracentrifugation, nitrogen cavitation is an excellent method to examine the shift of peripheral membrane proteins between cytosolic and membrane fractions.
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Registered trials
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