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, 410 (2), 313-5

An Assay to Measure the Affinity of Proteins for Microtubules by Quantitative Fluorescent Microscopy

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An Assay to Measure the Affinity of Proteins for Microtubules by Quantitative Fluorescent Microscopy

Beth Graczyk et al. Anal Biochem.

Abstract

We report a fluorescence-based assay for measuring the affinity of microtubule binding proteins for microtubules. The affinity of any fluorescently tagged protein for taxol-stabilized microtubules can be measured with this assay. We describe the assay and provide a detailed protocol. Using this assay, we found that the affinity of the Dam1 complex for microtubules is decreased by the presence of free unpolymerized tubulin and is sensitive to the salt concentration in the binding buffer. These effects may account for the previous differences in binding affinities reported.

Figures

Figure 1
Figure 1
Visualization of microtubule binding can distinguish between non-specific aggregation and microtubule binding. A. Dam1 complex bound to microtubules. Microtubule binding assay was performed as described except that the buffer conditions were BRB80 containing 33 mM NaCl, 3 mM HEPES, and 3 mM sodium phosphate. Green is Dam1 complex tagged with GFP. In this assay 10 nM Dam1 complex was incubated for 5 minutes with taxol-stabilized microtubules and then 2.5 nM unlabeled Ndc80 complex was added. B. Aggregates of the Dam1 complex. The Dam1 complex can form aggregates in the binding assay. In this assay, the order of addition was reversed. 2.5 nM unlabeled Ndc80 complex was incubated with taxol-stabilized microtubules for 5 minutes, and then 10 nM Dam1 complex was added.
Figure 2
Figure 2
Characterization of the microtubule binding assay. The microtubule binding assays were performed and quantified as described in the text except as noted below. A. Fixation prior to sedimentation gives the same result as fixation after sedimentation. After: Dam1 complex (2.5 or 5 nM) was incubated with taxol-stabilized microtubules (2.5 nM) for 10 minutes. The solution was not fixed. Instead, the bound Dam1 complex was separated from unbound by sedimentation through a 15% glycerol cushion made in BRB80 buffer. The supernatant and cushion were removed by aspiration and the coverslip was dipped in 2% glutaraldehyde before mounting on the slide. Before: Dam1 complex (2.5 or 5 nM) was incubated with taxol-stabilized microtubules for 10 minutes and then the solution was fixed before sedimentation through the glycerol cushion. The error bars represent standard error of the mean (SEM) for 4 replicates. B. The effect of glutaraldehyde concentration on measuring MT binding Dam1 complex (5 nM) was incubated with taxol-stabilized MTs (2.5 nM) for 10 minutes. An aliquot of glutaraldehyde (750 μl) was added to each assay tube (250 μl) and the samples were incubated for 15 min. The final concentrations of glutaraldehyde were as shown. The error bars represent the SEM for 4 replicates. C. The effect of fixation time on measuring MT binding Dam1 complex (5 nM) was added to taxol-stabilized MTs (3.2 nM) and incubated for 10 minutes. Samples were fixed for the given times. The error bars represent the SEM for 3 replicates. D. The effect of fixation volume on measuring MT binding Dam1 complex (5 nM) was added to taxol-stabilized MTs (2.5 nM) and incubated for 10 minutes. Different volumes of glutaraldehyde were added to each assay to yield the given ratio of volume of glutaraldehyde/volume of assay. The final concentration of glutaraldehyde was constant (1.5%). Error bars represent the SEM for 3–7 replicates. E. Time course of Dam1 complex binding to MTs. Dam1 complex (10 nM) was added to taxol-stabilized microtubules (2.5 nM). At the given times, glutaraldehyde was added to end the reaction. The error bars represent the SEM for 2–8 replicates. F. Non-specific binding was not detected Dam1 complex (filled circles) or GFP (open circles) (0.5–15 nM) were assayed. The data for the Dam1 complex are reproduced for comparison from [7]. They are plotted as a Hill plot [9] and the dotted line shows the fit to the McGhee and VonHippel model as described [7]. For the Dam1 complex the error bars represent the SEM for 8–11 replicates. The assays for binding GFP were done in quadruplicate. The SEM for the GFP data were smaller than the size of the symbols and are not shown. G. Dam1 complex binding to MTs is decreased by free tubulin. Dam1 complex binding to MTs was measured in the presence of the given quantities of unpolymerized tubulin (in nM). Unpolymerized tubulin in BRB80 containing 1 mM GTP was diluted 1/100 in BRB80 containing 10 μM taxol before addition to the assay. The error bars represent the SEM for 2–5 replicates. H. Dam1 complex binding to MTs is sensitive to salt. Dam1 complex (7.5 nM) was added to taxol-stabilized microtubules (2.5 nM) in BRB80 buffer containing the given concentration of NaCl. (Note that BRB80 is 120 mM K+). Error bars represent SEM for 3 replicates.

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