Tubulin polymerization conditions
Posted: Wed 29 Jun 2016 10:27Tubulin polymerization conditions
Technical Tip from CYTOSKELETON
Temperature
Tubulin polymerization is regulated by temperature. At 37°C tubulin will polymerize into microtubules while at 4°C microtubules will depolymerize to the tubulin subunits. There is a loss of 5% polymer per degree reduction in temperature. It is critical therefore to pay particular attention to temperature throughout the assay. Tubulin should be kept on ice until transferred to the 96 well plate for polymerization at 37°C. The plate reader should be brought to 37°C before beginning assay and the plate and buffers should be pre-warmed to 37°C.
Assay Characterization
In order to achieve reproducible results the researcher must decide on standard conditions of operation. The recommended standard conditions are 2-3 mg/ml tubulin in general tubulin buffer supplemented with 1 mM GTP and 15% glycerol. Using a higher protein concentration will achieve greater polymerization signal which can be useful for detecting inhibitors. Using lower or zero concentrations of glycerol is useful for detecting polymerization enhancing compounds. In the absence of glycerol, tubulin will not polymerize at a concentration below 5 mg/ml except in the presence of an enhancing agent like paclitaxel. Do not use high concentrations of taxol and glycerol together as this combination of enhancers causes aberrant tubulin polymer formations. Conditions can be modified to suit particular requirements. For example, if you wish to search for inhibitors that bind hydrophobic pockets of tubulin you may want to use no glycerol and a higher concentration of tubulin or use microtubule-associated proteins (MAPs; as in MAP-rich tubulin) which bind ionically rather than in hydrophobic pockets. Tubulin concentraton and glycerol concentration will also vary based on the purity of tubulin being used. Purer tubulin actually requies more enhancers than tubulin that also contains MAPs since MAPs are natural polymerization enhancers.
Technical Tip from CYTOSKELETON
Temperature
Tubulin polymerization is regulated by temperature. At 37°C tubulin will polymerize into microtubules while at 4°C microtubules will depolymerize to the tubulin subunits. There is a loss of 5% polymer per degree reduction in temperature. It is critical therefore to pay particular attention to temperature throughout the assay. Tubulin should be kept on ice until transferred to the 96 well plate for polymerization at 37°C. The plate reader should be brought to 37°C before beginning assay and the plate and buffers should be pre-warmed to 37°C.
Assay Characterization
In order to achieve reproducible results the researcher must decide on standard conditions of operation. The recommended standard conditions are 2-3 mg/ml tubulin in general tubulin buffer supplemented with 1 mM GTP and 15% glycerol. Using a higher protein concentration will achieve greater polymerization signal which can be useful for detecting inhibitors. Using lower or zero concentrations of glycerol is useful for detecting polymerization enhancing compounds. In the absence of glycerol, tubulin will not polymerize at a concentration below 5 mg/ml except in the presence of an enhancing agent like paclitaxel. Do not use high concentrations of taxol and glycerol together as this combination of enhancers causes aberrant tubulin polymer formations. Conditions can be modified to suit particular requirements. For example, if you wish to search for inhibitors that bind hydrophobic pockets of tubulin you may want to use no glycerol and a higher concentration of tubulin or use microtubule-associated proteins (MAPs; as in MAP-rich tubulin) which bind ionically rather than in hydrophobic pockets. Tubulin concentraton and glycerol concentration will also vary based on the purity of tubulin being used. Purer tubulin actually requies more enhancers than tubulin that also contains MAPs since MAPs are natural polymerization enhancers.