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

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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.

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