By Samols D., Agrawal A., Kushner I.
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The peak fractions are diluted threefold with buffer S: 20 mM 34 Kozasa Fig. 5. Ni-NTA column for purification of wild-type β1γ2 (A) or 6-His-β1γ2 (B) Fractions of 4 µL were subjected to SDS-PAGE and stained by silver nitrate. Lane 1, load; lane 2, flowthrough; lanes 3–5, wash with high salt and low imidazole; lanes 6–11, elution with AMF; lanes 12 and 13, elution with 150 mM imidazole. 7% CHAPS. The fractions are loaded onto Mono Q HR5/5 column that was equilibrated with buffer S. 7% CHAPS in buffer S can be replaced with 1% octylglucoside.
21. 5 g Coomassie Brilliant blue R-250, 600 mL H2O. 22. Coomassie blue destaining solution: 300 mL methanol, 100 mL glacial acetic acid, 600 mL H2O. 23. 0, 2 mM MgCl2, 1 mM EDTA. 24. Recombinant Gsα protein (27). 25. 1 mg/mL Pyruvate kinase (Roche Diagnostics, cat. no. 0128155, Indianapolis, IN). 26. 10 mM Forskolin in dimethylsulfoxide (DMSO). 27. 10 mM AlCl3. 28. 5 M NaF. 29. 32P-ATP (NEN, cat. no. com). 30. 3HcAMP (NEN, cat. no. com). 31. 75 mM cAMP. 32. 15 M potassium phosphenolpyruvate (Sigma-Aldrich, cat.