User:Brigette D. Black/Notebook/Brigettes Notebook/2009/06/17/Assay Attempt

Resuspending Kinesin
Cyctoskeleton recomends that it shoud be "reconstituted to 5 mg/ml with distilled water or CMW Buffer 1". The water resuspension would be the easiest, although since we are using BRB80 for the assay we probably want to use a buffer.

The buffer that they recommend, CMW, is given by:
 * 100mM PIPES (pH 7.0)
 * 200mM KCl
 * 2mM MgCl_2
 * 1mM DTT
 * 20 uM ATP

Instead, we are going to heat a little bit and use our own recipe. We will resuspend the kinesin in:


 * BRB80 (about 1mM)
 * BME (5mM)
 * KCl (200mM)
 * ATP (1mM)

With these concentrations I should add:


 * 1uL MgATP (at 100mM)
 * 20uL KCl (at 1M)
 * 0.5uL BME (at 100%)
 * 78.5uL BRB80
 * Total: 100 uL

Of this I need to pipette 5uL and add this to one tube of kinesin. Then very quickly put 1uL of kinesin into 5 aliquots, flash freeze, and then put into the -80 C freezer. Unfortunately, the cytoskeleton information tells us that kinesin will lose activity if kept in a concentration under that 5 mg/mL, so making aliquots at 5 uL/mg (one motility assay) does not seem to be a possibility. Also, the info sheet says that the kinesin should not be frozen and unfrozen repeatedly as it will also lose activity.

Today I mixed up a 1M KCl solution (pushed through a 0.2 um filter), so we have that made. Tomorrow I will combine all of the ingredients and mix it with the kinesin.