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.
