Nuclear Extract Preparation
Nuclear Extract Preparation - Related Videos
04:49
In Vitro Nuclear Assembly Using Fractionated Xenopus Egg Extracts
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Cited by 5 •
2008
Nuclear membrane assembly is an essential step in the cell division cycle; this process can be replicated in the test tube by combining Xenopus sperm chromatin, cytosol, and light membrane fractions. Complete nuclei are formed, including nuclear membranes with pore complexes, and these reconstituted nuclei are capable of normal nuclear processes.
07:56
Sample Preparation of Mycobacterium tuberculosis Extracts for Nuclear Magnetic Resonance Metabolomic Studies
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Cited by 8 •
2012
The metabolomic profile of Mycobacterium tuberculosis is determined after growth in broth cultures. Conditions can be varied to test the effects of nutritional supplements, oxidants, and anti-tuberculosis agents on the metabolic profile of this microorganism. Procedure for extract preparation is applicable for both 1D 1H and 2D 1H-13C NMR analyses.
08:05
Small-scale Nuclear Extracts for Functional Assays of Gene-expression Machineries
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Cited by 37 •
2012
A protocol for preparation of robust, small-scale HeLa nuclear extracts is described. This protocol is valuable for assays that require use of small populations of cells, such as cells treated with drugs or RNAi. The method should be applicable to a wide variety of gene expression assays and other cell types, including patient cells.
07:45
Preparation and Fractionation of Xenopus laevis Egg Extracts
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Cited by 7 •
2008
Crude and fractionated Xenopus egg extracts can be used to provide ingredients for reconstituting cellular processes for morphological and biochemical analysis. Egg lysis and differential centrifugation are used to prepare the crude extract which in turn in used to prepare fractionated extracts and light membrane preparations.
14:27
A Cell-Free Assay Using Xenopus laevis Embryo Extracts to Study Mechanisms of Nuclear Size Regulation
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Cited by 12 •
2016
Mechanisms of cellular and intra-cellular scaling remain elusive. The use of Xenopus embryo extracts has become increasingly common to elucidate mechanisms of organelle size regulation. This method describes embryo extract preparation and a novel nuclear scaling assay through which mechanisms of nuclear size regulation can be identified.
FAQs
Hypotonic conditions cause cells to swell, which promotes plasma-membrane rupture while helping nuclei remain intact. This selective disruption is essential because intact nuclei can then be separated from other cellular material by differential centrifugation. If nuclear integrity is not maintained, soluble nuclear proteins and DNA-binding complexes may become mixed with nonnuclear components, reducing the specificity of later analyses.
High-salt extraction changes the chemical environment around nuclear components and promotes the release of nuclear proteins, including transcription factors and DNA-binding complexes. Salt concentration must be controlled because insufficient extraction can limit recovery, whereas poorly controlled conditions may interfere with native protein interactions or activity. The resulting soluble fraction supports biochemical assays of nuclear function.
Temperature, protease inhibition, salt concentration, and mechanical disruption are central quality variables. Low-temperature handling and protease inhibitors help limit protein degradation, while controlled mechanical disruption supports nuclear integrity. Salt must be adjusted to release target proteins without unnecessarily disturbing functional interactions. Together, these conditions influence whether extracted proteins retain the activity or associations needed for analysis.
The process begins by disrupting cells under hypotonic conditions, followed by differential centrifugation to separate intact nuclei from other cellular material. The isolated nuclear fraction then undergoes controlled high-salt extraction to release soluble nuclear proteins and complexes. Careful handling during disruption, separation, and extraction helps produce a fraction suitable for downstream biochemical measurements.
Temperature should be carefully controlled throughout preparation, and protease inhibition should be included to help preserve nuclear proteins. These safeguards are particularly important when the extract will be tested for DNA-binding, enzymatic, or transcriptional activity. Maintaining protein integrity improves the likelihood that assay results reflect the original nuclear components rather than degradation introduced during sample handling.
Nuclear extracts can be used in electrophoretic mobility shift assays to examine DNA-binding complexes, immunoblotting to detect specific proteins, enzyme assays to assess nuclear enzymatic activity, and in vitro transcription studies to investigate transcriptional function. In biology research, these applications connect nuclear components with gene regulation, signaling, and chromatin-associated activity.