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Related Experiment Videos

Isolation of nuclear envelopes with polyanions.

M Bornens, J C Courvalin

    The Journal of Cell Biology
    |January 1, 1978
    PubMed
    Summary

    Heparin effectively isolates pure nuclear envelopes from cell nuclei with high yield. Optimal conditions involve specific salt concentrations and temperatures, preserving envelope structure and composition for biochemical analysis.

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    Area of Science:

    • Cell Biology
    • Molecular Biology
    • Biochemistry

    Background:

    • Nuclear envelopes are crucial for cellular function, regulating molecular traffic between the nucleus and cytoplasm.
    • Efficient isolation methods are needed to study nuclear envelope structure and composition.
    • Heparin has shown potential in nuclear envelope isolation, but optimal conditions require definition.

    Purpose of the Study:

    • To establish optimal conditions for isolating nuclear envelopes using heparin.
    • To perform morphological and biochemical characterization of heparin-isolated nuclear envelopes.
    • To investigate the roles of specific ions and alternative polyanions in the isolation process.

    Main Methods:

    • Incubation of isolated nuclei with heparin under varying conditions (temperature, time, salt concentrations).
    • Isolation of nuclear envelopes via sedimentation and sucrose density gradient analysis.
    • Biochemical analysis of isolated nuclear envelopes for protein, phospholipid, RNA, and DNA content.
    • Morphological examination of isolated nuclear envelopes.

    Main Results:

    • Optimal isolation achieved with 40 min heparin incubation at 4°C, yielding nearly 100% pure nuclear envelopes.
    • Isolated envelopes retained perinuclear space and outer leaflet ribosomes; buoyant density of 1.18.
    • Chemical composition: 62% proteins, 34% phospholipids, 3% RNA, 0.5% DNA.
    • Low sodium phosphate (2-10 mM) critical for chromatin solubilization; Mg++ counteracted association; EDTA disrupted pore visibility, suggesting Ca++/Mg++ dependence.

    Conclusions:

    • Heparin provides an efficient and advantageous method for isolating pure nuclear envelopes.
    • Optimal isolation conditions preserve structural integrity and allow for detailed biochemical analysis.
    • The study elucidates the critical roles of specific ions and demonstrates heparin's superiority over other polyanions for this purpose.

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