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Tubulin Polymerisation Assays

Introduction

Tubulin polymerizes to form microtubules in a dynamic process that is important for many aspects of cell regulation, division and motility. Tubulin dynamics (polymerization versus depolymerization rates) during cell division are also a major target for drug discovery.

Turbidometric Format

Microtubules scatter light and hence polymerization kinetics can be measured by spectroscopic absorbance at 340 nm. The bovine tubulin containing versions (Cat nr BK004 and BK006) have been replaced with the porcine versions (cat. nr BK004P and BK006P). These kits provide all reagents and detailed instructions necessary to perform polymerization assays.

Kit BK006P uses >99% pure tubulin for polymerization measurements and generates a highly robust signal (Figure 1 in the gallery). BK006 is recommended for the study and characterization of microtubule associated proteins (MAPs) and measurement of IC50 and EC50 values for MAPs and tubulin binding compounds.

Kit BK004P uses >97% tubulin for primary screen polymerization measurements. The signal generated by BK004P is approximately 50% that of BK006, it also has a higher coefficient of variation and is less senitive to modulators of tubulin dynamics. However, BK004P is more economical than BK006P and hence is a cost effective method for assay set-up, primary screens and initial characterization of tubulin binding compounds.

Fluorescence Based Tubulin Polymerization Assays (Cat nr BK011P & BK011)

Porcine tubulin based fluorescence polymerization assay (Cat nr BK0011P). This assay is the same as the bovine tubulin kit (Cat nr BK011) except for the source of tubulin.
Both kits measure tubulin polymerization as a function of enhanced fluorescence of a reporter molecule during the polymerization process (Figure 2 in the gallery). The great advantage of these assays is that they allow miniaturization of the reaction, something that is not easily achievable with the traditional turbidometric based assay.
The assays are recommended in situtations utilizing purified cancer cell line, plant cell or fungal tubulins, where tubulin protein is a limiting factor or in high throughput screens.

Comparison to the fluorescence format

The turbidometric format assays are traditionally the standard way to measure tubulin polymerization (Lee and Timascheff 1978). The fluorescent format is more sensitive and more cost effective. Cf Table 1 in the gallery for comparison of the two formats.