CytoPhos™ Phosphate Assay (1-500ug/ml protein reactions)

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The Cytophos phosphate assay Biochem Kit is an extremely quick and economical way to measure phosphate in solution. There is minimal interference from other common ions and reagents used in molecular biology and biochemistry. The manual is designed to guide you through the process of measuring end-point and kinetic assays. There is special consideration for ATPase and GTPase assays, which can be performed easily with this kit.
The CytoPhos phosphate assay is specifically developed for measuring phosphate in lower protein concentration solutions (<0.5 mg/ml). For measuring phosphate in high protein concentration solutions, Cytoskeleton, Inc. recommends using the PhosFree phosphate assay (Cat. # BK050).

Question 1: What is the sensitivity of the CytoPhos Phosphate Assay Biochem Kit (Cat. #BK054)?

Answer 1: The ATPase assay is sensitive over a range of 1 &mu, M &ndash, 50 &mu, M Pi (equivalent to 0.1 nmoles &ndash, 5 nmoles Pi in 100 &mu, l reaction volume).

Question 2: If assay optimization is necessary with the CytoPhos Phosphate Assay Biochem Kit (Cat. #BK054), what should I focus on?

Answer 2: The ATPase end-point assay kit has been developed to provide a good general substrate for a broadrange of ATPase proteins, however, there are several parameters that may affect ATPase activity, andthese include:

Protein concentration. A titration of the ATPase of interest should be performed to achieve optimal results. This assay is suitable for protein concentrations below 1 mg/ml. If higher protein concentrations are required, the PhosFree Phosphate Assay Kit (Cat. # BK050) should be considered.

Reaction buffer conditions. Salt concentration (e.g., 20 mM-1000 mM) and pH should be titrated for optimal activity.

ATP concentration. To minimize background readings, an ATP concentration of 0.3 - 0.6 mM is recommended. An ATP titration should be performed to obtain optimal results.

Control Reactions. It is important to include control reactions in the assay, particularly if the ATPase of interest is in an impure state.

Half Area Well Plates.We recommend the use of a half area well plate (180 &mu, l volume) to perform the assays, as this will maintain the pathlength while allowing smaller reaction volumes to be used. The protocols for this assay will describe reactions for a 100 &mu, l final volume. If standard 300 &mu, l volume wells are to be used, we recommend using a 200 &mu, l final volume.

500

Spectrophotometer capable of measuring absorbance at 650 nm wavelength.
Small capacity (100-1000 ul) cuvettes or 96-well microtiter plates.