Dynamin GTPase assay

For 20ul reactions (enough for >30 timepoints):

1. Mix together 2ul 10x reaction buffer (1250mM NaCl, 50mM KCl, 200mM Hepes, 10mM MgCl2) with 1-2ul lipid tubes (1mg/ml), make up volume with water.

2. Add dynamin to give 0.1-1.0 uM final conc. Incubate for at least 10min at RT.

3. Add GTP spiked with 32P GTP. Usually final conc. will be between 50uM and 1mM. Start timer immediately on addition and mixing of the GTP.

4. Spot 0.5-0.7ul of reaction onto Polygram CEL 300 PEI/UV254 (Sigma) TLC plates at desired timepoints. At very short (<1min.) timepoints it may be necessary to stop reactions once spotted onto the plate using a high temp. hair drier.

5. After the last spots have dried run the TLC plate at least 2/3 of the way in 0.75M KH2PO4 pH3.5.

6. Dry the TLC plate in a warm air drier or such-like and autorad or phosphorimage.

Tubule, Dyn and GTP concentrations may all be varied.


NB: Merck PLC plates 20x20cm PEI cellulose F (1.05579) with buffer 1M Acetic Acid (stock=17.47) + 0.8M LiCl separates the GTP from GDP better.