Cytosolic Fraction Isolation
Cytosolic Fraction Isolation - Related Videos
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.
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.
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).
10:03
Isolation of CA1 Nuclear Enriched Fractions from Hippocampal Slices to Study Activity-dependent Nuclear Import of Synapto-nuclear Messenger Proteins
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Cited by 8 •
2014
We provide a detailed protocol for induction of long-term potentiation in the CA1 region of the hippocampus and the subsequent isolation of nuclear enriched fractions from the tetanized area of the slice. This approach can be used to determine activity dependent nuclear protein import in cellular models of learning and memory.
08:22
Preparation of Mitochondrial Enriched Fractions for Metabolic Analysis in Drosophila
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Cited by 13 •
2015
Mitochondria play central roles in the regulation of metabolism and homeostasis. Subtle changes in mitochondrial metabolism that affect organismal physiology could be difficult to detect in whole organism metabolomics studies. Here we describe an isolation method that enhances the detection of subtle metabolic shifts in Drosophila melanogaster.
FAQs
Differential centrifugation separates cellular material according to whether it forms a pellet under successive centrifugation conditions. After gentle lysis, nuclei and organelles sediment, whereas soluble cytosolic components remain in the supernatant. This physical separation reduces interference from membrane-bound and organelle-associated material, allowing subsequent measurements to focus more specifically on soluble intracellular components.
Gentle lysis releases cellular contents while helping preserve the soluble proteins, metabolites, and signaling molecules intended for analysis. Excessively disruptive conditions could increase unwanted structural material in the sample or compromise component integrity, reducing interpretability. Controlling lysis conditions therefore supports a cleaner separation and improves the reproducibility of measurements made from the resulting supernatant.
The retained material primarily includes soluble cytosolic proteins, metabolites, and signaling molecules. Nuclei, organelles, membranes, and other cellular structures are separated into pellets during the centrifugation workflow. This distinction matters because it lets investigators examine soluble processes with less contribution from other cellular compartments, although the quality of separation depends on carefully controlled processing conditions.
A typical workflow begins by gently lysing the cells, then applying differential centrifugation to sediment nuclei and organelles. The soluble material is collected from the supernatant after the relevant pelleting steps. Researchers can then analyze this fraction for properties such as enzyme activity, protein expression, post-translational regulation, or intracellular signaling, depending on the biological question.
Lysis conditions, temperature, and centrifugation steps require careful control. Together, these factors influence whether cellular structures separate effectively and whether soluble cytosolic components remain suitable for analysis. Consistent handling across samples helps preserve the targeted material and supports reproducible comparisons, which is especially important when evaluating changes in protein expression, enzyme activity, or signaling.
Cytosolic fraction isolation is useful when a study needs to focus on soluble intracellular events rather than the combined contents of the whole cell. The recovered fraction can support investigations of enzyme activity, protein expression, post-translational regulation, and signaling molecules. By reducing contributions from membranes and organelles, the approach helps relate measured changes more directly to cytosolic cellular function.