User:Steven J. Koch/Notebook/Kochlab/2009/06/23/Kinesin bulk activity assays

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Steve Koch 16:03, 23 June 2009 (EDT): We received two bulk (ensemble) ATPase assay kits from cytoskeleton today:

  • CytoPhos Phosphate Assay BIOCHEM KIT BK054
  • PhosFree Phosphate Assay Biochem Kit (Cat. #BK050)

The PhosFree assay is the one I'm familiar with from my postdoc work. During that time, I got to the point where I was pretty good at doing time course assays, and for sure if we do a ton of them, it will be cheaper to work from scratch (purchasing malachite green powder, etc.). On the other hand, the powder is nasty and tough to work with, so for not high throughput, I bet using this kit is better. Furthermore, I could never get very linear standard curves (even though the original Gilbert papers seemed to show very nice standard curves), so potentially the kit will work better.

I also ordered the CytoPhos, because I wasn't sure which kit we would like better, and so I ordered both for the sake of expedience and learning.

CytoPhos PhosFree (Malachite Green)
Range 1 to 15 μM phosphate (0.1 nmoles to 1.5 nmoles) 20 to 1000 μM phosphate (if doing a single endpoint, aim for 500 micromolar)
Volume 30 microliter reaction volume, 100 microliter final volume 20 microliter reaction volume, 220 microliter recommended final volume
Wavelength 650 nm 650 nm
Protein concentration Below 1 mg / ml
Other time sensitive color development; less sensitive to Pi contamination (probably); more steps
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