Standard Fluorescence Motility Assay

Materials

Standard Solutions

BRB80 =

80 mM PIPES pH 6.85

1 mM MgCl₂

1 mM EGTA

BRB80T =

BRB80
10 micromolar Taxol^*

BRB80CS0.5 =

BRB80

0.5 mg/mL Casein

BRB80CA =

BRB80

0.2 mg/mL Casein

1 mM MgATP

BRB80CT =

BRB80

0.2 mg/mL Casein

10 micromolar Taxol^*

^*Add Taxol (1 mM in DMSO) while vortexing solution

Kinesin (0.05 – 5 micrograms/mL diluted in BRB80CA)

Whatman #4 Filter Paper or KimWipes

Procedure

1.	Load 20 microliters of BRB80CS0.5 into the flow cell and let stand for 5 min. Then load 20 microliters of kinesin into the flow cell by wicking through with Whatman #4 filter paper or a KimWipe, and let stand for 5 min. Wash two times with 50 microliters BRB80CA using the filter paper or a KimWipe to draw through. Then add 20 microliters of motility solution.
2.	Observe using microscope with 100X objective, 25°C, 1X optivar, and low viscosity immersion oil. Record microtubule motility onto VHS or S-VHS tape.

Notes

1.	Make stock solutions (GTP, DMSO, MgATP, Taxol, etc.) ahead of time, divide into 10 microliter aliquots and store at -20°C. For use, thaw 1 aliquot and keep on ice.
2.	Make new motility solution after 1.5 hours. Antifade will die after this time.
3.	Keep MT100, motility solution, Taxol, and BRB80CT at room temperature. Keep others on ice.
4.	Make sure casein is soluble and in the right concentration. This is the cause of most problems. Also make sure the BME that is used is good.

Contributed by William Hancock, J. Howard Laboratory

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Created 11April 1999 22:10 GMT

Modified 27 March 2002 19:05 GMT