Abstract
G13 belongs to the G12-subfamily of heterotrimeric regulatory G-proteins. Employing specific antibodies, we isolated Gα13 from bovine brain by a four-step purification protocol combining conventional and affinity chromatography. The use of ethylene glycol as a protective agent influenced the elution properties of Gα13 markedly. Only in the presence of ethylene glycol (30% v/v) a clear separation of Gα13 from other G-proteins was achieved during the initial anion exchange chromatography. This allowed isolation of Gα13 by subunit exchange chromatography on βγ-agarose. Gα13 was only released from immobilized βγ-dimers via activation by AMF but not by GTPγS, pointing to a poor basal nucleotide exchange of this protein. In contrast, N-terminally truncated Gα13 did not bind to immobilized βγ-dimers.
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