In this study, we have used a Mg2+-specific fluorescent probe (furaptra) to probe Mg2+ binding to Gsα and Giα1 proteins, and identified two Mg2+ q vd oph in both the inactive GDP-bound and active GTPγS (a non-hydrolyzable GTP analog)-bound conformations.
Materials and methods
G protein expression and purification. Giα1 and Gsα proteins containing N-terminal hexahistidine tags were isolated from BL21(.DE3) Escherichiacoli cells harboring the expression vectors for these proteins by the method of Lee et al. [19]. The crude protein mixture obtained was equilibrated with Ni2+–NTA agarose resin (Qiagen) in 50 mM Tris–Cl, pH 8.0, containing 10 mM imidazole and 10 mM β-mercaptoethanol before being poured into a gravity column. The Ni2+–NTA agarose resin was washed with 50 mM Tris–Cl, pH 8.0, containing 20 mM imidazole, 250 mM NaCl, and 10 mM β-mercaptoethanol, and the desired protein was eluted with 50 mM Tris–Cl, pH 8.0, containing 150 mM imidazole and 10 mM β-mercaptoethanol. Isolated G proteins were further purified using an FPLC with an ion exchange Q-sepharose column. The presence and purity (>97%) of isolated protein was confirmed by SDS–PAGE.
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Immunohistochemistry and assessment Antibodies used were as follows IL goat
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