FEBS Letters | |
Affinity labeling of GTP‐binding proteins in cellular extracts | |
Faulhammer, Heinz G1  Sprinzl, Mathias1  Löw, Andreas1  | |
[1] Laboratorium für Biochemie, Universität Bayreuth, Postfach 10 12 51, D-8580 Bayreuth, Germany | |
关键词: GTP-binding protein; Affinity labeling; Peroxidase oxidation; NKXD; Consensus sequence; HEPES; (N-[2-hydroxyethyl]piperazine-N′-[2-ethane-sulfonic acid]); Tricine; (N-tris[hydroxymethyl]-methylglycine; SDS-PAGE; SDS-polyacrylamide gel electrophoresis; | |
DOI : 10.1016/0014-5793(92)80478-Y | |
学科分类:生物化学/生物物理 | |
来源: John Wiley & Sons Ltd. | |
【 摘 要 】
GTP-binding proteins in cellular extracts from Escherichia coli, Thermus thermophilus, yeast, wheat germ or calf thymus were identified using in situ periodate-oxidized [α-32P]GTP as affinity label. Site-specific reaction of individual GTP-binding proteins was achieved by cross-linking the protein-bound 2′,3′-dialdehyde derivative of GTP with the single lysine residue of the conserved NKXD sequence through Schiff's base formation and subsequent cyanoborohydride reduction. Labeled GTP-binding proteins from prokaryotic or eukaryotic cell homogenates were separated by polyacrylamide gel electrophoresis and visualized by autoradiography. In addition cross-linking of [α-32P]GTP with GTP-binding proteins was demonstrated in model systems using different purified GTPases, human c-H-ras p21, transducin from bovine retina, polypeptide elongation factor Tu (EF-Tu) from T. themophilus and initiation factor 2 (1F2) from T. thermophilus. The described affinity labeling technique can serve as an analytical method for the identification of GTPases belonging to the classes of ras-proteins, elongation and initiation factors, and heterotrimeric signal transducing G-proteins.
【 授权许可】
Unknown
【 预 览 】
Files | Size | Format | View |
---|---|---|---|
RO201912020296346ZK.pdf | 541KB | download |