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. Author manuscript; available in PMC: 2010 Jun 30.
Published in final edited form as: Biochemistry. 2009 Jun 30;48(25):6002–6011. doi: 10.1021/bi900602h

Figure 2. Microtubule binding of pseudo-phosphorylation mutants.

Figure 2

The microtubule binding affinities of wild-type and mutant tau were measured using a centrifugal assay to separate bound and unbound tau. A) A representative SDS-PAGE gel after centrifugation. The concentrations of standard tau in first five lanes are 0.06, 0.125, 0.25, 0.5 and 1 μg, respectively. Tau protein, concentration varying from 0 to 10 μM, was mixed with microtubules at constant total MT concentration of 1.62 μM tubulin dimers. Bound tau was separated from free tau by centrifugation and fractionated on the SDS-PAGE lane 6–15. B) Two representative curves plotting the concentration of bound tau versus tau free in solution (● wild-type tau, ○ 7-Phos tau). The amount of bound tau was determined by the intensity of tau band on SDS-PAGE. The data were fit to a one site binding equation. Kd and n were determined for each of three independent repetitions of the binding curves for each protein and shown in panels C and D, respectively. All data are presented as the average of three independent determinations of Kd and n ± s.d. (n=3). Values that were significantly different from wild-type values at P < 0.05 (*) and P < 0.01 (**) were determined by t test.