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Related Experiment Videos

Characterization of nuclear matrices prepared without salt extraction.

B S Dwarakanath1, C A Wallen, D K St Clair

  • 1Department of Radiology, Bowman Gray School of Medicine of Wake Forest University, Winston-Salem, North Carolina 27103.

Analytical Biochemistry
|October 1, 1991
PubMed
Summary

Nuclear matrices prepared using proteases (PRT-matrices) were analyzed. Pronase-treated matrices closely resemble high-salt matrices, making them suitable for studying nuclear matrix-associated DNA.

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Area of Science:

  • Cell Biology
  • Biochemistry

Background:

  • Nuclear matrices are structural components of the cell nucleus.
  • Conventional preparation methods (high-salt extraction) can interfere with certain biochemical analyses.

Purpose of the Study:

  • To characterize protease-prepared nuclear matrices (PRT-matrices).
  • To compare PRT-matrices with high-salt matrices (HS-matrices).
  • To identify PRT-matrices suitable for salt-sensitive studies.

Main Methods:

  • DNase I and protease digestion (Type XIV protease/pronase) for PRT-matrix preparation.
  • Protein and DNA gel electrophoresis.
  • Flow cytometry and scanning electron microscopy.

Main Results:

  • Pronase-treated PRT-matrices maintained structural integrity.

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  • PRT-matrices had minimal associated nuclear DNA (<5%).
  • PRT-matrices lacked higher-order chromatin structure and had DNA fragments of 400-1100 bp.
  • PRT-matrices showed reduced protein content compared to HS-matrices.

    • Conclusions:

    • Protease-prepared nuclear matrices, particularly those treated with pronase, are comparable to HS-matrices.
    • PRT-matrices are suitable for studying nuclear matrix-associated DNA properties.
    • PRT-matrices may not be ideal for investigating specific nuclear matrix-associated proteins.