Abstract
Chicken erythroid nuclei were prepared using four published methods. Our findings indicate that nuclei prepared by nitrogen cavitation are less likely to be contaminated with plasma membrane fragments than those made by procedures involving cell disruption by hypotonic lysis. However, globin gene sequences were much less sensitive to DNase I digestion in nuclei prepared by nitrogen cavitation. This suggests that the conformation of chromatin was altered by the cavitation procedure. Analysis of the proteins solubilized during limited DNase I digestion of nuclei prepared by both hypotonic lysis and cavitation revealed no appreciable differences in HMG proteins but a notable difference in the RNP-associated proteins and core histones.
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Abbreviations
- HMG:
-
high mobility group nonhistone chromosomal protein
- RNP:
-
ribonucleoprotein
- SSC:
-
14 mM sodium citrate buffered saline pH 7.0
- PMSF:
-
phenylmethanesulfonyl fluoride
- EDTA:
-
ethylenediaminetetraacetic acid
- DTT:
-
dithiothreitol
- PBS:
-
10 mM sodium phosphate buffered saline pH 7.2
- NP-40:
-
Nonidet P-40 (octylphenoxypolyethoxyethanol)
- SS-DNA:
-
single-stranded DNA
- RSB:
-
reticulocyte standard buffer, 0.01 M NaCl, 0.003 M MgCl2, 0.01 M Tris-HCI, pH 7.4.
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Briggs, J.A., Montiel, M.M., Briggs, R.C. et al. Comparison of chicken erythroid cell nuclear isolation methods using morphological, immunochemical and biochemical criteria. Mol Cell Biochem 74, 29–42 (1987). https://doi.org/10.1007/BF00221910
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DOI: https://doi.org/10.1007/BF00221910