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. Author manuscript; available in PMC: 2014 Mar 24.
Published in final edited form as: Biochem J. 2011 Mar 15;434(3):415–426. doi: 10.1042/BJ20101437

Figure 6. SPR-based equilibrium-binding analysis of the affinity for PtdIns(3,4,5)P3 of two PBD1 mutants.

Figure 6

Two arginine residues in PBD1 (that correspond with Arg399 and Arg417 in PPIP5K1) were mutated to alanine and the constructs R399A (at 45, 70, 120, 200, 300, 375, 850 and 1700 nM) (A and B) and R417A (at 150, 300, 400, 600, 700, 1100, 1600 and 1900 nM) (C and D) were injected at 5 μl/min over immobilized PtdCho/PtdE/PtdIns(3,4,5)P3 vesicles and the resulting changes in the RU are plotted as a function of time to yield the SPR-binding sensorgrams (A and C), from which the Kd values were determined (B and D). The buffer contained 100 mM KCl, 6 mM MgCl2, 5 mM Na2 ATP and 25 mM Hepes (pH 7.2).