Cytosol Fractionation
Cytosol Fractionation - Related Videos
10:41
Detection of Toxin Translocation into the Host Cytosol by Surface Plasmon Resonance
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Cited by 14 •
2012
In this report, we describe how surface plasmon resonance is used to detect toxin entry into the host cytosol. This highly sensitive method can provide quantitative data on the amount of cytosolic toxin, and it can be applied to a range of toxins.
08:08
Measurement of Cytosolic Ca2+ in Isolated Contractile Lymphatics
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Cited by 24 •
2011
We introduce an approach to evaluate the cytosolic Ca2+ concentration in isolated lymphatics to study Ca2+-dependent and Ca2+-sensitizing mechanisms of lymphatic smooth muscle contraction.
13:55
Measurement of Vacuolar and Cytosolic pH In Vivo in Yeast Cell Suspensions
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Cited by 40 •
2013
Vacuolar and cytosolic pH can be measured in live yeast (S. cerevisiae) cells using ratiometric fluorescent dyes localized to specific cellular compartments. We describe procedures for measuring vacuolar pH with BCECF-AM, which localizes to the vacuole in yeast, and cytosolic pH with a cytosolic ratiometric pH-sensitive GFP (yeast pHluorin).
09:25
Quantification of Cytosolic vs. Vacuolar Salmonella in Primary Macrophages by Differential Permeabilization
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Cited by 19 •
2015
We describe the quantification of cytosolic and vacuolar Salmonella typhimurium in bone-marrow derived macrophages using differential digitonin permeabilization.
06:27
An Assay to Quantify Cytosolic and Vacuolar Salmonella in Infected Primary Macrophages
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2025
This video demonstrates an assay to quantify cytosolic and vacuolar Salmonella typhimurium inside bone marrow-derived macrophages using differential permeabilization. The macrophages are infected with a fluorescent protein-expressing Salmonella, which is internalized inside a specialized vacuolar structure called Salmonella-containing vacuole or SCV. A subset of bacteria escape the SCV and enter the cytosol. The cholesterol-rich plasma membrane of macrophages is permeabilized using digitonin,...
FAQs
Differential centrifugation separates components according to how readily they sediment under progressively higher centrifugal forces. Larger or denser structures, including nuclei, mitochondria, membrane fragments, and other particles, form sediment at earlier or stronger separation steps. Soluble proteins and metabolites remain in the supernatant, allowing investigators to collect a fraction enriched in soluble cytoplasmic contents for subsequent analysis.
Controlled cell lysis releases cytoplasmic contents while helping preserve the molecules being studied. If handling disrupts the intended separation or damages native molecules, the resulting fraction may no longer accurately represent their original biochemical state. Maintaining careful conditions therefore supports more reliable measurements of protein localization, enzyme activity, signaling pathways, metabolism, and molecular interactions.
The cytosolic fraction is the soluble portion recovered in the supernatant after particulate structures have sedimented. Removed material includes nuclei, mitochondria, membrane fragments, and other cellular structures. This distinction matters because analyzing the supernatant separately reduces the structural complexity of the sample and focuses measurements on soluble proteins and metabolites rather than mixed cellular contents.
Fractionation quality directly influences whether measured molecules can be associated confidently with the soluble cytoplasmic compartment. Incomplete separation may leave particulate material in the supernatant, while poor handling may alter native molecules. Careful preparation and centrifugation improve the accuracy of downstream biochemical, molecular, and proteomic assays by producing a more appropriate sample for the intended analysis.
A typical workflow begins by lysing cells under controlled conditions, then subjecting the lysate to differential centrifugation. Successive centrifugation steps sediment nuclei, mitochondria, membrane fragments, and other particles at progressively higher forces. The supernatant containing soluble cytoplasmic contents is retained as the cytosolic fraction and directed to the selected downstream assay.
The isolated material can support measurements of protein localization, enzyme activity, signaling pathways, metabolism, and interactions with cellular components. Because membrane-bound and organelle-associated structures are separated from soluble contents, the fraction provides a more focused sample for biochemical, molecular, or proteomic assays. The appropriate analysis depends on the biological question and the molecules being examined.
This method is useful when researchers need to examine soluble cytoplasmic contents without analyzing the entire cellular mixture at once. It can clarify where proteins are localized, characterize soluble enzyme activity, investigate signaling or metabolic processes, and assess interactions involving cytoplasmic components. The resulting fraction therefore connects cell organization with focused biochemical and molecular measurements.