Nuclear Protein Extraction
Nuclear Protein Extraction - 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.
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.
05:43
A Protein Preparation Method for the High-throughput Identification of Proteins Interacting with a Nuclear Cofactor Using LC-MS/MS Analysis
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Cited by 1 •
2017
We have established a method for the purification of coregulatory interaction proteins using the LC-MS/MS system.
10:05
Visualization of Protein-protein Interaction in Nuclear and Cytoplasmic Fractions by Co-immunoprecipitation and In Situ Proximity Ligation Assay
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Cited by 15 •
2017
Protein-protein interactions can occur in both the nucleus and the cytoplasm of a cell. To investigate these interactions, traditional co-immunoprecipitation and modern proximity ligation assay are applied. In this study, we compare these two methods to visualize the distribution of NF90-RBM3 interactions in the nucleus and the cytoplasm.
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
Sequential lysis separates two structural barriers. First, a mild buffer disrupts the plasma membrane while preserving intact nuclei. Researchers can then isolate the nuclear material before applying stronger detergent or high-salt treatment. This order helps focus subsequent analysis on proteins released from the nuclear compartment, including soluble and chromatin-associated components.
After nuclei have been isolated, stronger detergent or high-salt treatment disrupts the nuclear envelope and releases material that milder lysis leaves contained. The resulting extract can include both soluble proteins and proteins associated with chromatin. This distinction matters when examining transcription factors, histones, or other nuclear components whose recovery depends on disrupting the nuclear compartment.
The method can provide access to soluble nuclear proteins as well as chromatin-associated proteins. These populations include transcription factors, histones, and other nuclear components identified in the extract. Examining them through Western blotting, immunoprecipitation, or DNA-binding assays helps connect nuclear protein analysis with gene regulation, chromatin organization, and nuclear signaling.
The workflow begins with mild lysis to disrupt the plasma membrane while keeping nuclei intact. Researchers then isolate the nuclei and apply a stronger detergent or high-salt treatment to disrupt the nuclear envelope. The resulting nuclear extract can be analyzed by Western blotting, immunoprecipitation, or DNA-binding assays, depending on the biological question.
Nuclear protein extracts can be analyzed by Western blotting, immunoprecipitation, or DNA-binding assays. These readouts allow investigators to examine selected nuclear proteins, investigate protein-associated material, or study DNA-binding behavior. The resulting evidence supports research on transcription factors, histones, gene regulation, chromatin organization, and nuclear signaling.
Its applications extend across cell biology, developmental research, disease-mechanism studies, and drug-response investigations. By focusing analysis on nuclear proteins, researchers can examine components connected with gene regulation, chromatin organization, and nuclear signaling in these contexts. The approach is particularly relevant when nuclear components are central to the biological question being investigated.