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. Key features of PiColorLock™ - phosphate detection reagent 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
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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- Potent in vitro Interference of Fleroxacin in DNA-binding, Unwinding and
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- Bcl-2 associated athanogene 5 (Bag5) is overexpressed in prostate cancer and
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- P. gingivalis-Nucleoside-diphosphate-kinase Inhibits ATP-Induced Reactive-Oxygen-Species via P2X7 Receptor / NADPH-Oxidase Signaling and Contributes to Persistence - Cellular Microbiology (2012)
- Novel 2, 4-Pyrimidine Derivatives And Use Thereof - European Patent (2012)
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- Common Pathogenic Effects of Missense Mutations in the P-Type ATPase ATP13A2 (PARK9) Associated with Early-Onset Parkinsonism - PLOS One (2012)
- Structure of the VirB4 ATPase, alone and bound to the core complex of a type IV secretion system - PNAS (2012)
- Peroxisomal proteostasis involves a Lon family protein that functions as protease and chaperone - JBC (2012)
- Xeroderma pigmentosum group F protein binds to Eg5 and is required for proper mitosis: implications for XP-F and XFE - Genes to Cells (2012)
- hSWS1·SWSAP1 Is an Evolutionarily Conserved Complex Required for Efficient Homologous Recombination Repair - JBC (2011)
- Enzyme Stability of Microencapsulated Bifidobacterium animalis ssp. lactis Bb12 after Freeze Drying and during Storage in Low Water Activity at Room Temperature - Journal of Food Science (2011)
- Digitoxin and a synthetic monosaccharide analog inhibit cell viability in lung cancer cells - Toxicology and Applied Pharmacology (2011)
- Functional defect caused by the 4544G>A SNP in ABCC2: potential impact for drug cellular disposition - Pharmacogenetics and Genomics (2011)
- A Common Variant in TFB1M Is Associated with Reduced Insulin Secretion and Increased Future Risk of Type 2 Diabetes - Cell Metabolism (2011)
- Overexpression of the Endoplasmic Reticulum Chaperone BiP3 Regulates XA21-Mediated Innate Immunity in Rice – PLoS One (2010)
- Identification of gp96 as a Novel Target for Treatment of Autoimmune Disease in Mice - PLoS One (2010)
- Antimicrobial Mechanism of Action of Transferrins: Selective Inhibition of H+-ATPase - Antimicrob Agents Chemother. (2010)
- Modulation of UvrD Helicase Activity by Covalent DNA-Protein Cross-links - Biol Chem. (2010)
- Monoclonal antibodies against nucleoside triphosphate hydrolase-II can reduce the replication of Toxoplasma gondii - Parasitology (2010)
- Biochemical Dissection of the ATPase TraB, the VirB4 Homologue of theEscherichia coli pKM101 Conjugation Machinery - J. Bacteriol. (2010)
- Identification of gp96 as a Novel Target for Treatment of Autoimmune Disease in Mice – PLoS One (2010)
- ATP-binding cassette-like transporters are involved in the transport of lignin precursors across plasma and vacuolar membranes - PNAS (2010)
- Arabidopsis stromal 70-kDa heat shock proteins are essential for chloroplast development - Planta (2010)
- Lipopeptide induces apoptosis in fungal cells by a mitochondria-dependent pathway - Peptides (2010)
- Modulation of a Schistosoma mansoni multidrug transporter by the antischistosomal drug praziquantel - The FASEB Journal (2010)
- Biosynthesis of (Bacterio)chlorophylls ATP-Dependent Transient Subunit Interaction And Electron Transfer Of Dark Operative Protochlorophyllide Oxireductase - JBC (2010)
- Stress-Dependent Coordination of Transcriptome and Translatome in Yeast - PLoS Biology (2009)
- Crystal Structure of a Novel Conformational State of the Flavivirus NS3 Protein: Implications for Polyprotein Processing and Viral Replication - J Virol (2009)
- ATP and MO25? Regulate the Conformational State of the STRAD? Pseudokinase and Activation of the LKB1 Tumour Suppressor – PLoS Biol (2009)
- Apratoxin A Reversibly Inhibits the Secretory Pathway by Preventing Cotranslational Translocation - Molecular Pharmacology (2009)
- NS4A regulates the ATPase activity of the NS3 helicase: a novel cofactor role of the non-structural protein NS4A from West Nile virus - Journal of General Virology (2009)
- Evidence That Two ATP-Dependent (Lon) Proteases in Borrelia burgdorferi Serve Different Functions - PLoS Pathogens (2009)
- ATP and MO25? Regulate the Conformational State of the STRAD? Pseudokinase and Activation of the LKB1 Tumour Suppressor - PLoS Biology (2009)
- Stress-Dependent Coordination of Transcriptome and Translatome in Yeast - PLoS Biology (2009)
- An Afg2/Spaf-Related Cdc48-like AAA ATPase Regulates the Stability and Activity of the C. elegans Aurora B Kinase AIR-2 - Developmental Cell (2008)
- Staphylococcus aureus HrtA Is an ATPase Required for Protection against Heme Toxicity and Prevention of a Transcriptional Heme Stress Response - Journal of Bacteriology (2008)
- ATP scavenging by the intracellular pathogen Porphyromonas gingivalis inhibits P2X7-mediated host-cell apoptosis - Cell Microbiol. (2008)
- The Two-component NS2B-NS3 Proteinase Represses DNA Unwinding Activity of the West Nile Virus NS3 Helicase - JBC (2008)
- Functional characterization of the nucleotide binding domain of the Cryptosporidium parvum CpABC4 transporter: An iron–sulfur cluster transporter homolog - Molecular and Biochemical Parasitology (2008)
- Hippocampal levels of gamma-enolase, C-1-tetrahydrofolate synthase and serotransferrin fluctuate over the estrous cycle in the rat - Neuroscience (2008)
- Consumption of Soy Protein Isolate Modulates the Phosphorylation Status of Hepatic ATPase/ATP Synthase ß Protein and Increases ATPase Activity in Rats - J. Nutr. (2007)
- First identification and functional characterization of an immunogenic protein in unculturable haemotrophic Mycoplasmas (Mycoplasma suis HspA1) - FEMS Immunology & Medical Microbiology (2007)
- Identification, structure and mode of action of a new regulator of the Helicobacter pylori HP0525 ATPase - EMBO J. (2007)
- Proteome Profiling in the Rat Harderian Gland - J Proteome Res (2006)