High Throughput Colorimetric GTPase Assays (inc. PiColorLock™)

This non radioactive colorimetric assay kit contains all the necessary reagents for measuring enzyme activity (everything included in the PiColorlock™ reagent 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 GTPases 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).

Kit Components:

Components in the 2-plate GTP 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 GTP (10 vials)
  • Store at room temperature
  • 2 x 96-well plates (5 plates)

 

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The data below is for the PiColorLock™ reagent which is included in GTPase assay kits.

Figure 1. Our PiColorLock has also been designed to have a large linear range, thus reducing the need for sample dilution. Competitors’ products are linear over a much narrower range of concentrations.

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Frequently Asked Questions (FAQs) for PiColorLock Phosphate Detection System

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.

Which reagents does the PiColorLock Phosphate Detection System contain?

Reagent Product Code: 303-0030 Product Code: 303-0125
PiColorLock 30ml 125ml
Stabilizer 27.5ml 55ml
Accelerator 1ml 2ml
Phosphate Standard 5ml 11ml

Please see here for ordering & shipping information.

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