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Alterations in the phenotype of plant cells studied by NH2-terminal amino acid-sequence analysis of proteins electroblotted from two-dimensional gel-separated total extracts

Guy Bauw UGent, Marc De Loose UGent, Dirk Inzé UGent, Marc Van Montagu UGent and Joël Vandekerckhove (1987) PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA. 84(14). p.4806-4810
abstract
Phenotypic alterations induced by the cytokinin 6-benzylaminopurine in cell suspensions of Nicotiana plumbaginifolia were studied at the level of the NH(2)-terminal sequence of the constituent proteins. Total protein extracts were separated by classical two-dimensional PAGE, and the proteins were recovered by electroblotting onto support materials allowing direct gas-phase sequence analysis of the immobilized proteins. The systems used consist of an efficient electrotransfer buffer (50 mM Tris borate, pH 8.3) in combination with either glass-fiber sheets to which poly(4-vinyl-N-methylpyridinium iodide) is adsorbed or with membranes of polyvinylidene difluoride. The former is an improved version of our previously reported Polybrene-coated glass-fiber sheets and was found to be at least twice as efficient as the polyvinylidene difluoride blots. Thirteen proteins were selected for analysis. They were either induced, repressed, or independent of cytokinin. Ten proteins yielded a sequence, ranging from 10 to 38 residues. Three of the studied Nicotiana proteins show a degree of homology higher than 85% with the amino acid sequences of other eukaryotic proteins-triose-phosphate isomerase, Mn superoxide dismutase, and (1,3)-beta-glucanase. The latter enzyme was repressed by the plant hormone. This study demonstrates that proteins associated with phenotypic variations in cells can now be sequenced by a straightforward procedure involving two-dimensional gel separation of total cellular proteins, recovery by electroblotting, and gas-phase sequence analysis of the immobilized proteins.
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author
organization
year
type
journalArticle (original)
publication status
published
subject
journal title
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
Proc. Natl. Acad. Sci. USA
volume
84
issue
14
pages
4806 - 4810
Web of Science type
Article
ISSN
0027-8424
DOI
10.1073/pnas.84.14.4806
language
English
UGent publication?
yes
classification
A1
copyright statement
I have transferred the copyright for this publication to the publisher
id
1902278
handle
http://hdl.handle.net/1854/LU-1902278
date created
2011-09-16 15:30:24
date last changed
2016-12-19 15:41:56
@article{1902278,
  abstract     = {Phenotypic alterations induced by the cytokinin 6-benzylaminopurine in cell suspensions of Nicotiana plumbaginifolia were studied at the level of the NH(2)-terminal sequence of the constituent proteins. Total protein extracts were separated by classical two-dimensional PAGE, and the proteins were recovered by electroblotting onto support materials allowing direct gas-phase sequence analysis of the immobilized proteins. The systems used consist of an efficient electrotransfer buffer (50 mM Tris borate, pH 8.3) in combination with either glass-fiber sheets to which poly(4-vinyl-N-methylpyridinium iodide) is adsorbed or with membranes of polyvinylidene difluoride. The former is an improved version of our previously reported Polybrene-coated glass-fiber sheets and was found to be at least twice as efficient as the polyvinylidene difluoride blots. Thirteen proteins were selected for analysis. They were either induced, repressed, or independent of cytokinin. Ten proteins yielded a sequence, ranging from 10 to 38 residues. Three of the studied Nicotiana proteins show a degree of homology higher than 85\% with the amino acid sequences of other eukaryotic proteins-triose-phosphate isomerase, Mn superoxide dismutase, and (1,3)-beta-glucanase. The latter enzyme was repressed by the plant hormone. This study demonstrates that proteins associated with phenotypic variations in cells can now be sequenced by a straightforward procedure involving two-dimensional gel separation of total cellular proteins, recovery by electroblotting, and gas-phase sequence analysis of the immobilized proteins.},
  author       = {Bauw, Guy and De Loose, Marc and Inz{\'e}, Dirk and Van Montagu, Marc and Vandekerckhove, Jo{\"e}l},
  issn         = {0027-8424},
  journal      = {PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA},
  language     = {eng},
  number       = {14},
  pages        = {4806--4810},
  title        = {Alterations in the phenotype of plant cells studied by NH2-terminal amino acid-sequence analysis of proteins electroblotted from two-dimensional gel-separated total extracts},
  url          = {http://dx.doi.org/10.1073/pnas.84.14.4806},
  volume       = {84},
  year         = {1987},
}

Chicago
Bauw, Guy, Marc De Loose, Dirk Inzé, Marc Van Montagu, and Joël Vandekerckhove. 1987. “Alterations in the Phenotype of Plant Cells Studied by NH2-terminal Amino Acid-sequence Analysis of Proteins Electroblotted from Two-dimensional Gel-separated Total Extracts.” Proceedings of the National Academy of Sciences of the United States of America 84 (14): 4806–4810.
APA
Bauw, G., De Loose, M., Inzé, D., Van Montagu, M., & Vandekerckhove, J. (1987). Alterations in the phenotype of plant cells studied by NH2-terminal amino acid-sequence analysis of proteins electroblotted from two-dimensional gel-separated total extracts. PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 84(14), 4806–4810.
Vancouver
1.
Bauw G, De Loose M, Inzé D, Van Montagu M, Vandekerckhove J. Alterations in the phenotype of plant cells studied by NH2-terminal amino acid-sequence analysis of proteins electroblotted from two-dimensional gel-separated total extracts. PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA. 1987;84(14):4806–10.
MLA
Bauw, Guy, Marc De Loose, Dirk Inzé, et al. “Alterations in the Phenotype of Plant Cells Studied by NH2-terminal Amino Acid-sequence Analysis of Proteins Electroblotted from Two-dimensional Gel-separated Total Extracts.” PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA 84.14 (1987): 4806–4810. Print.