PiColorLock™ Phosphate Detection System
PiColorLock™ phosphate detection reagent
PiColorLock™ phosphate detection reagent changes colour in the presence of inorganic phosphate (Pi). This can be exploited to measure any enzyme that generates Pi including ATPases, GTPases, phosphatases, heat-shock proteins and DNA unwinding proteins.
The unique formulation of PiColorLock™ affords enhanced assay linearity, dynamic range and colour stability; it is also possible with PiColorLock™ to work with unstable substrates (e.g. ATP, GTP) that give high non-enzymatic background signals with other acidic dye-based detection reagents.
PiColorLock™ - phosphate detection reagent features and benefits
|Colorimetric assay||Simple, scalable and non-radioactive|
|Compatible with almost any assay buffer||Flexibility over a wide range of existing assays|
|Stable reagent formulation||Long shelf life|
|Unique accelerator||Speeds up color development|
|Unique stabilizer||Suppresses non enzymatic backgrounds with acid-labile substrates (ATP or GTP)|
|PiColorLock™-Pi complex is very stable||No precipitation, results can be measured over several hours – ideal for HTS|
|Wide linear range||No inhibition of color development by high concentrations of protein|
For more information on PiColorLock™ take a look at our Phosphate Detection Guide.
For more information, please watch our webinar on drug screening-assays for phosphate-generating enzymes below:
Please do not hesitate to Contact us to find out more!
Figure 1. 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).
Figure 2. ATP has been incubated in three detection reagents. A steadily rising background signal is seen with competitor reagents, whereas PiColorLock™ gives baseline readings.
Figure 3. 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.
Which reagents does the PiColorLock™ Phosphate Detection System contain?
|Reagent||Product Code: 303-0030||Product Code: 303-0125|
- The membrane remodeling protein Pex11p activates the GTPase Dnm1p during peroxisomal fission - PNAS (2015)
- Crystal structure of the Bloom's syndrome helicase indicates a role for the HRDC domain in conformational changes - Nucleic Acids Research (2015)
- Protein-pyridinol thioester precursor for biosynthesis of the organometallic acyl-iron ligand in [Fe]-hydrogenase cofactor - Nature Communications (2015)
- Development of a mitochondria-targeted Hsp90 inhibitor based on the crystal structures of human TRAP1 - Journal of the American Chemical Society (2015)
- Functional analysis of TPM domain containing Rv2345 of Mycobacterium tuberculosis identifies its phosphatase activity - Protein Expression and Purification (2015)
- Preparation and characterization of human ADCK3, a putative atypical kinase - Protein Expression and Purification (2014)
- A continuous enzyme-coupled assay for triphosphohydrolase activity of HIV-1 restriction factor SAMHD1 - Antimicrobial Agents and Chemotherapy (2014)
- Assays, Surrogates, and Alternative Technologies for a TB Lead Identification Program Targeting DNA Gyrase ATPase - Journal of Biomolecular Screening (2014)
- Optimization of Pyrrolamides as Mycobacterial GyrB ATPase Inhibitors: Structure-Activity Relationship and In Vivo Efficacy in a Mouse Model of Tuberculosis - Antimicrobial Agents and Chemotherapy (2014)
- Allosteric Activation of Bordetella pertussis Adenylyl Cyclase by Calmodulin: Molecular Dynamics and Mutagenesis Studies - Journal of Biological Chemistry (2014)
- Self-Assembly of MinE on the Membrane Underlies Formation of the MinE-Ring to Sustain Function of the E. coli Min System - Journal of Biological Chemistry (2014)
- Quantification of biliary excretion and sinusoidal excretion of 5(6)-carboxy-2?,7?-dichlorofluorescein (CDF) in cultured hepatocytes isolated from Sprague Dawley, Wistar and Mrp2-deficient Wistar (TR?) rats - Toxicology in Vitro (2014)
- The PIKE Homolog Centaurin gamma Regulates Developmental Timing in Drosophila - PLoS One (2014)
- Low Molecular Weight Protein Tyrosine Phosphatases Control Antibiotic Production in Streptomyces coelicolor A3(2) - Enzyme Engineering (2014)
- Ablation of Osteopontin Improves the Skeletal Phenotype of Phospho1-/- Mice - Journal of Bone and Mineral Research (2014)
- Prospecting for Unannotated Enzymes: Discovery of a 3?,5?-Nucleotide Bisphosphate Phosphatase within the Amidohydrolase Superfamily - Biochemistry (2013)
- Mutations in the Chromodomain-like Insertion of Translation Elongation Factor 3 Compromise Protein Synthesis Through Reduced ATPase Activity - Journal of Biological Chemistry (2013)
- Bordetella pertussis adenylate cyclase toxin translocation across a tethered lipid bilayer - PNAS (2013)
- 3,4-Methylenedioxy-?-nitrostyrene inhibits NLRP3 activation by blocking assembly of the inflammasome - Journal of Biological Chemistry (2013)
- A biosensor of local kinesin activity reveals roles of PKC and EB1 in KIF17 activation - Journal of Cell Biology (2013)
- RNA silencing of genes involved in Alzheimer's disease enhances mitochondrial function and synaptic activity - Biochimica et Biophysica Acta (2013)
- Chrysophaentins are competitive inhibitors of FtsZ and inhibit Z-ring formation in live bacteria - Bioorganic & Medicinal Chemistry (2013)
- A novel cholesterol-producing Pichia pastoris strain is an ideal host for functional expression of human Na,K-ATPase ?3?1 isoform - Applied Microbiology and Biotechnology (2013)
- Direct regulation of microtubule dynamics by KIF17 motor and tail domains - Journal of Biological Chemistry (2013)
- Extracellular ATP Released by Osteoblasts Is A Key Local Inhibitor of Bone Mineralisation - PLoS One (2013)
- Plastid division control: the PDV proteins regulate DRP5B dynamin activity - Plant Molecular Biology (2013)
- Structural and functional characterization of a phosphatase domain within yeast general transcription factor TFIIIC - The Journal of Biological Chemistry (2013)
- ATP-dependent transport of statins by human and rat MRP2/Mrp2 - Toxicology and Applied Pharmacology (2013)
- Characterisation of ATP-Dependent Mur Ligases Involved in the Biogenesis of Cell Wall Peptidoglycan in Mycobacterium tuberculosis - PLoS One (2013)
- A calcineurin docking motif (LxVP) in dynamin-related protein 1 contributes to mitochondrial fragmentation and ischemic neuronal injury - The Journal of Biological Chemistry (2013)
- Na+ Regulation in the Malaria Parasite Plasmodiumfalciparum Involves the Cation ATPase PfATP4 and Is a Target of the Spiroindolone Antimalarials - Cell Host & Microbe (2013)
- A Newly Uncovered Group of Distantly Related Lysine Methyltransferases Preferentially Interact with Molecular Chaperones to Regulate Their Activity - PLoS Genetics (2013)
- Polyphosphates inhibit extracellular matrix mineralization in MC3T3-E1 osteoblast cultures - Bone (2013)
- Structural and Mechanistic Characterization of L-Histidinol Phosphate Phosphatase from the PHP Family of Proteins - Biochemistry (2013)
- Mesenchymal Stem Cell Therapy - Humana Press (2013)
- Kinetic Modelling of GlmU Reactions – Prioritization of Reaction for Therapeutic Application - PLoS One (2012)
- The unusual mycobacterial chaperonins: evidence for in vivo oligomerization and specialization of function - Molecular Microbiology (2012)
- Synthesis and Antibacterial Evaluation of a New Series of N-Alkyl-2-alkynyl/(E)-alkenyl-4-(1H)-quinolones - Molecules (2012)
- PPM1H Is a p27 Phosphatase Implicated in Trastuzumab Resistance - Cancer Discovery (2011)
- Sepsis impairs alveolar epithelial function by downregulating Na-K-ATPase pump - AJP - Lung Physiol (2011)
- Structural and functional insights into DNA-end processing by the archaeal HerA helicase–NurA nuclease complex - Nucleic Acids Research (2011)
- Sepsis impairs alveolar epithelial function by downregulating Na-K-ATPase pump - AJP - Lung Physiol (2011)
- FXYD Proteins Stabilize Na,K-ATPase, Amplification Of Specific Phosphatidylserine -Protein Interactions - JBC (2011)
- Structural and functional insights into DNA-end processing by the archaeal HerA helicase–NurA nuclease complex - Nucl Acids Res (2011)
- Stabilization of the ?2 isoform of Na,K-ATPase by mutations in a phospholipid binding pocket - JBC (2011)
- Characterisation of multiple substrate-specific (d)ITP/(d)XTPase and modelling of deaminated purine nucleotide metabolism - MIMS EPrints (2011)
- Substrate uptake, phosphorus repression, and effect of seed culture on glycopeptide antibiotic production: Process model development and experimental validation - Biotechnology and Bioengineering (2010)
- Selectivity of Digitalis Glycosides for Isoforms of Human Na,K-ATPase - JBC (2010)
- ATP-dependent MurE ligase in Mycobacterium tuberculosis: Biochemical and structural characterisation - Tuberculosis (2010)
- Mechanism of Action of an Imidopiperidine Inhibitor of Human Polynucleotide Kinase/Phosphatase - JBC (2010)
- Sensor combination and chemometric variable selection for online monitoring of Streptomyces coelicolor fed-batch cultivations - Applied Microbiology and Biotechnology (2010)
- Roles of Electrostatics and Conformation in Protein-Crystal Interactions - PLoS One (2010)
- Anti-tubercular screening of natural products from Colombian plants: 3-methoxynordomesticine, an inhibitor of MurE ligase of Mycobacterium tuberculosis - J Antimicrob Chemother (2010)
- Oligomeric assembly and interactions within the human RuvB-like RuvBL1 and RuvBL2 complexes - Biochem. J. (2010)
- Functional study on GTP hydrolysis by the GTP-binding protein from Sulfolobus solfataricus, a member of the HflX family - J Biochem (2010)
- Identification of a Small Molecule Inhibitor of the Human DNA Repair Enzyme Polynucleotide Kinase/Phosphatase - Cancer Res (2009)
- Structural and Mutational Analyses of Deinococcus radiodurans UvrA2 Provide Insight into DNA Binding and Damage Recognition by UvrAs - Structure (2009)
- Characterization of Streptococcus gordonii SecA2 as a Paralogue of SecA - J Bacteriol (2009)
- Characterization of Human GTPBP3, a GTP-Binding Protein Involved in Mitochondrial tRNA Modification - Mol Cell Biol (2008)
- Stabilization of Na+,K+-ATPase Purified from Pichia pastoris Membranes by Specific Interactions with Lipids - Biochemistry (2007)
- Purification of the Human ?2 Isoform of Na,K-ATPase Expressed in Pichia pastoris. Stabilization by Lipids and FXYD1 - Biochemistry (2007)
- Benzimidazole covalent probes and the gastric H+/K+-ATPase as a model system for protein labeling in a copper-free setting - Molecular BioSystems (2016)
- Structure-Based Mutagenesis of Sulfolobus Turreted Icosahedral Virus B204 Reveals Essential Residues in the Virion-Associated DNA-Packaging ATPase - J. Virol. (2016)
- Insight into Structure-Function Relationships and Inhibition of the Fatty Acyl-AMP Ligase (FadD32) Orthologs from Mycobacteria - J. Biol. Chem. (2016)
- Embryo defective 14 encodes a plastid-targeted cGTPase essential for embryogenesis in maize - Plant J. (2015)
- Concerted and differential actions of two enzymatic domains underlie Rad5 contributions to DNA damage tolerance - Nucleic Acids Research (2015)
- Digoxin derivatives with selectivity for the α2β3 isoform of Na,K-ATPase potently reduce intraocular pressure - Proc Natl Acad Sci USA (2015)
- Characterization of the Catalytic and Nucleotide Binding Properties of the Alpha-kinase domain of Dictyostelium Myosin-II Heavy Chain Kinase A. - The Journal of Biological Chemistry (2015)
- Independent mechanisms target SMCHD1 to H3K9me3-modified chromatin and the inactive X chromosome - Molecular and Cellular Biology (2015)
- Structure of the Dictyostelium Myosin-II Heavy Chain Kinase A (MHCK-A) [alpha]-kinase domain apoenzyme reveals a novel autoinhibited conformation - Scientific Rep. (2016)
- Reduced Dynamin-related Protein 1 Protects Against Phosphorylated Tau-induced Mitochondrial Dysfunction and Synaptic Damage in Alzheimer's Disease - Oxford Journals (2016)
- Selective assembly of Na,K-ATPase α2β2 heterodimers in the heart: distinct functional properties and isoform-selective inhibitors - J Biol Chem (2016)
- Development of a one-pot assay for screening and identification of Mur pathway inhibitors in Mycobacterium tuberculosis - Nature (2016)
- Structural and functional studies of the Leishmania braziliensis mitochondrial Hsp70: Similarities and dissimilarities to human orthologues - Arch Biochem Biophys. (2016)