Isolation of active DNA-binding nuclear proteins from tomato galls induced by root-knot nematodes

We describe 2 methods for extraction of DNA-binding proteins from root-knot nematode feeding sites (ie, galls). DNA-binding activity was assayed by electrophoretic mobility shift assays using fragments from the root-knot nematode-responsive LEMMI9 and 35S promoters. In noninfected tissue, the method...

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Bibliographic Details
Published in:Plant molecular biology reporter 2001-12, Vol.19 (4), p.375-376
Main Authors: Escobar, Carolina, Aristizéabal, Fabio, Navas, Alfonso, Del Campo, Francisca F, Fenoll, Carmen
Format: Article
Language:English
Subjects:
Assaying
Deoxyribonucleic acid
DNA
DNA-binding protein
DNA-binding proteins
Electrophoretic mobility
Fragments
Galls
Nematodes
nuclear proteins
Nuclei
Promoters
Proteins
root-knot nematodes
Roots
Tomatoes
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Summary:We describe 2 methods for extraction of DNA-binding proteins from root-knot nematode feeding sites (ie, galls). DNA-binding activity was assayed by electrophoretic mobility shift assays using fragments from the root-knot nematode-responsive LEMMI9 and 35S promoters. In noninfected tissue, the method based on nuclei enrichment through a Percoll cushion was superior for isolation of DNA-protein binding activity with both promoters. With infected roots; the method based on crude extracts performed better with the LEMMI9 promoter, whereas nuclei-enriched extracts worked better with the 35S promoter. Therefore, both methods can be used to extract proteins for DNA-binding assays from infected roots, but the method of choice may depend on the promoter under study.
ISSN:0735-9640
1572-9818
DOI:10.1007/BF02772837