User:Brigette D. Black/Notebook/Brigettes Notebook/2009/08/31/CytoPhos Phosphate Assay

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After doing so much collection and analysis with the PhosFree Phosphate assay from Cytoskeleton, I decided it was probably about time to crack open the CytoPhos assay and see how the two compare. The Cytophos assay is supposed to be better at measuring low concentrations of phosphate in solution, while the PhosFree is better for larger concentrations. From what I could gather, the CytoPhos assay is more sensitive (more malachite green perhaps?) and thus small differences have a greater impact on the absorbances, while the PhosFree is less sensitive, so large concentrations are not all indistinguishably black.

I made the standard curve for the assay by using 0.1 mM Phosphate Standard which I made by diluting the 1mM phosphate Standard that had already been prepared. I then pipetted the following amounts into cuvettes:

  • Cuvette 1: Blank
  • Cuvette 2: 7.5 uL (6.25 uM)
  • Cuvette 3: 15 uL (12.5 uM)
  • Cuvette 4: 22.5 uL (18.75 uM)
  • Cuvette 5: 30 uL (25 uM)
  • Cuvette 6: 37.5 uL (31.25 uM)
  • Cuvette 7: 45 uL (37.5 uM)
  • Cuvette 8: 52.5 uL (43.75 uM)
  • Cuvette 9: 60 uL (50 uM)

I then pipetted Di H2O into the pipettes to bring the total for each to 120 uL. I then added 280 uL of the Reagent Solution, the amounts for these were based off of the manual sent by Cytoskeleton. The Reagent Solution, from my best guess, is a combination of perchloric acid (or another strong acid) and malachite green.

The user manuals between the two assays was very different as to how the concentration of phosphate was determined. The Cytophos manual describes a total amount molar amount of phosphate per 100 uL of total volume. The other kit defines concentration as the concentration of phosphate before any additional solution (except of water) is added. This did pose some difficulty in trying to relate the two. I did the math incorrectly about several times, but did finally do it correctly and came up with the concentrations in parenthesis above.

I measured each cuvette after 16 minutes and found the following plot.


The linear fit above shows that the absorbances at this concentration grow very linearly, so inferring phosphate concentrations will be very easy. In comparison with the PhosFree assay we can see that the absorbance for low concentration values (< 50 uM) is higher and thus is more sensitive and could give more accurate readings for low phosphate concentrations, such as with in the kinetics assay.