Colorimetric assays for GTPases are invariably based on the formation of colored complexes between an inorganic phosphate and a dye molecule under acidic conditions. Such assays are beset with problems of reagent precipitation and high backgrounds caused by impure substrates (i.e. contaminated with inorganic phosphate) and/or by non-enzymatic (acid) hydrolysis of the substrate.
At the heart of our assay kits is PiColorLock Gold, a superior phosphate detection reagent which ensures high stability of the colored dye-phosphate complexes. Together with specially purified substrates and proprietary stabilizers our kits offer the lowest possible assay backgrounds and outstanding assay performance.
Uniquely, Innova Biosciences GTPase assay kits contain highly purified GTP, which have had any free inorganic phosphate removed - this means that assays have the lowest possible background signal.
PiColorLock Phosphate Detection Systems can also be purchased for developing simple assays for any phosphate-generating enzyme.
This non radioactive colorimetric assay kit uses a 96 well format, and all the necessary reagents are supplied for measuring enzyme activity and are ideal for high throughput drug screening.
Colorimetric Assay Kit
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FAQs for GTPase (6)
May we have the protocol for 602-0120?The info. from http://www.novusbio.com/support/protocols/colorimetric-gtpase-assay-kit-protocol.html seems insufficient.
Here is the corresponding full protocol for your customer for this kit. I hope they find this information useful. http://www.innovabiosciences.com/images/stories/innova/pdfs/gtpase_assay_system_protocol_r3.pdf
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.
I have 5% DMSO in my assay. Can I use PiColorLock Gold?
Yes, the reagent is designed for drug screening work and other situations that require DMSO.
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 stabilizer with the sample and detection reagent. Make sure the stabilizer 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 Gold Phosphate Detection System for this purpose?
Yes, only the phosphate will give a signal; pyrophosphate will not.
I was wondering if any of the Lightning-Link kit components contain phosphates, and whether you can tell me the concentration?
The Lightning-Link kits do contain phosphate. The final concentration will vary depending on the amount of antibody you add to the vial, but should always be less than 15mM.