High Throughput Colorimetric ATPase Assays (inc. PiColorLock)
This non radioactive colorimetric assay kit contains all the necessary reagents for measuring enzyme activity (everything included in the PiColorlock kit and more) and is ideal for high throughput drug screening.
The kits contain PiColorlock™, a non-radioactive, superior phosphate detection reagent. Key features of PiColorLock™ include:
- Colorimetric Assay – competitor assays are radioactive.
- Special additive ensures low backgrounds with acid-labile substrates.
- Unparalleled stability of phosphate-dye complexes.
- Reagent is compatible with almost any assay buffer.
- No inhibition of color development by high concentrations of protein.
- Stable reagent formulation - long shelf life.
Colorimetric assays for ATPases are invariably based on the formation of colored complexes between an inorganic phosphate and a dye molecule under acidic conditions.
Competitor assays are beset with several problems including reagent precipitation. The high stability of PiColorLock™ ensures high stability of the colored dye-phosphate complexes (green color- see below).
Components in the 2-plate ATP assay kit (5-plate kit amounts in brackets)
Store at 4°C:
1 x 10ml of PiColorLock™* (1 x 25ml)
1 x 0.25ml of Accelerator (1 x 0.5ml)
1 x 5ml of Stabiliser (1 x 10ml)
1 x 1.5ml of 0.1M MgCl2 (2 x 1.5ml)
1 x 5ml of 0.5M Tris pH 7.5 (1 x 10ml)
1 x 5ml of 0.1mM Pi standard (1 x 10ml)
*Take care, the solution is very acidic
Store at -20 or -70oC
4 vials of lyophilized ATP (10 vials)
Store at room temperature
2 x 96-well plates (5 plates)
Figure 1. ATP has been incubated in three detection reagents. A steadily rising background signal is seen with competitor reagents, whereas PiColorLock™ gives baseline readings.
Figure 2. PiColorLock has also been designed to have a large linear range, thus reducing the need for sample dilution. For the purposes of comparison, data for PiColorLock ALS reagent (no longer available) is also shown, as it provides similar absorbance values to competitor products, at least at low levels of inorganic phosphate. Competitors’ products are linear over a much narrower range of concentrations.
I have a high background in my ATPase assay and I definitely do not have free phosphate in my sample
This is almost always due to inadequate mixing of the special stabiliser with the sample and detection reagent. Make sure the stabiliser is pipetted up and down several times to ensure thorough mixing.
I would like to measure the conversion of pyrophosphate to phosphate. Can I use the PiColorLock Phosphate Detection System for this purpose?
Yes, only the phosphate will give a signal; pyrophosphate will not.
I have 5% DMSO in my assay. Can I use PiColorLock?
Yes, the reagent is designed for drug screening work and other situations that require DMSO.
I have phosphate in my enzyme. What can I do?
You can dialyse or desalt the enzyme into a phosphate-free buffer. Alternatively, you can use a special resin (PiBind) to remove the phosphate.
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