GTPase Activity Kit (Colorimetric)

Product Discontinued

GTPase Activity Kit (Colorimetric) Summary

• Description: 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 our PiColorlock, a non-radioactive, superior phosphate detection reagent.
  
  Key Features of PiColorLock:
  • 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.
• Kit Type: Activity Kit (Colorimetric)

Applications/Dilutions

Publications

Read Publications using 602-0120 in the following applications: EnzAct 2 publications Func 1 publication In vitro 1 publication

Packaging, Storage & Formulations

• Storage: Storage of components varies. See protocol for specific instructions.

Notes

This product is manufactured by Abcam and distributed by Novus Biologicals.

This product is for research use only and is not approved for use in humans or in clinical diagnosis. This product is guaranteed for 1 year from date of receipt and this statement overrides any mentioned guarantee period on the limitations section of this products datasheet. Please contact technical@novusbio.com with questions.

Background

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 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 reagent may also be purchased separately for developing simple assays for any phosphate-generating enzyme. It is offered in both high (Gold) and standard sensitivity (ALS) formulations to suit almost any assay situation.

Limitations

This product is for research use only and is not approved for use in humans or in clinical diagnosis. Kits are guaranteed for 6 months from date of receipt.

Publications for GTPase Kit (602-0120)(40)

Publications using 602-0120

• Sarkar C, Sadhukhan T, Bagh MB et al. Cln1-mutations suppress Rab7-RILP interaction and impair autophagy contributing to neuropathology in a mouse model of infantile neuronal ceroid lipofuscinosis J. Inherit. Metab. Dis. 2020-04-12 [PMID: 32279353]
• Cherubini M, Puigdellivol M, Alberch J, Gines S et al. Cdk5-mediated mitochondrial fission: A key player in dopaminergic toxicity in Huntington's disease Biochim. Biophys. Acta. 2015-07-02 [PMID: 26143143] (Mouse)
• Yan J, Liu XH, Han MZ et al. Blockage of GSK3 beta-mediated Drp1 phosphorylation provides neuroprotection in neuronal and mouse models of Alzheimer’s disease. Neurobiol Aging 2014-01-01 [PMID: 25192600]
• Li W, Feng J, Gao C et al. Nitration of Drp1 provokes mitophagy activation mediating neuronal injury in experimental autoimmune encephalomyelitis Free Radical Biology and Medicine 2019-08-01 [PMID: 31377418] (EnzAct)
• Ferreira JCB, Campos JC, Qvit N et al. A selective inhibitor of mitofusin 1-beta IIPKC association improves heart failure outcome in rats. Nat Commun. 2019-04-09 [PMID: 30659190] (In vitro)
• Hoque A, Sivakumaran P, Bond ST et al. Mitochondrial fission protein Drp1 inhibition promotes cardiac mesodermal differentiation of human pluripotent stem cells. Cell Death Discov. 2018-12-01 [PMID: 29531836] (Human)
• Lin SJ, Chiang MC, Shih HY et al. Regulator of G protein signaling 2 (Rgs2) regulates neural crest development through Ppard-Sox10 cascade. Biochim. Biophys. Acta. 2016-12-13 [PMID: 27979767] (Func, Fish)
• Franco A, Kitsis RN, Fleischer JA et al. Correcting mitochondrial fusion by manipulating mitofusin conformations. Nature. 2016-12-01 [PMID: 27775718]
• Ho DH, Jang J, Joe EH et al. G2385R and I2020T Mutations Increase LRRK2 GTPase Activity. Biomed Res Int. 2016-01-01 [PMID: 27314038]
• Hu Y, Keniry M, Palmer SO, Bullard JM. Discovery and Analysis of Natural Product Compounds Inhibiting Protein Synthesis in Pseudomonas aeruginosa. Antimicrob. Agents Chemother. 2016-05-31 [PMID: 27246774]
• Yin X, Manczak M, Reddy PH. Mitochondria-targeted Molecules MitoQ and SS31 Reduce Mutant Huntingtin-induced Mitochondrial Toxicity and Synaptic Damage in Huntington's Disease. Hum. Mol. Genet. 2016-02-16 [PMID: 26908605]
• Hwang J, Kim HS, Kang BS et al. RGS19 converts iron deprivation stress into a growth-inhibitory signal. Biochem Biophys Res Commun. 2015-08-14 [PMID: 26116529]
• Cha GH, Wang W, Peng T et al. A Rac1 GTPase is a critical factor in the immune response of shrimp (Litopenaeus vannamei) to Vibrio alginolyticus infection. Dev Comp Immunol. 2015-01-01 [PMID: 25892021]
• Ellinger D, Glockner A, Koch J et al. Interaction of the Arabidopsis GTPase RabA4c with Its Effector PMR4 Results in Complete Penetration Resistance to Powdery Mildew. Plant Cell 2014-01-01 [PMID: 25056861]
• Gundner AL, Hahn I, Sendscheid O et al. The PIKE Homolog Centaurin gamma Regulates Developmental Timing in Drosophila. PLoS One 2014-01-01 [PMID: 24845618]
• Santhosh KT, Sikarwar AS, Hinton M et al. Thromboxane Receptor Hyper-Responsiveness in Hypoxic Pulmonary Hypertension Requires Serine 324. Br J Pharmacol. 2013-01-01 [PMID: 24490858]
• Alaganan A, Fentress SJ, Tang K et al. Toxoplasma GRA7 effector increases turnover of immunity-related GTPases and contributes to acute virulence in the mouse. PNAS 2013-01-01 [PMID: 24390541]
• Palmer SO, Rangel EY, Hu Y et al. Two Homologous EF-G Proteins from Pseudomonas aeruginosa Exhibit Distinct Functions. PLoS One. 2013-11-08 [PMID: 24260360]
• Zhang K, Fishel Ben Kenan R, Osakada Y et al. Defective Axonal Transport of Rab7 GTPase Results in Dysregulated Trophic Signaling. J Neurosci 2013-01-01 [PMID: 23616551]
• Qi X, Qvit N, Su YC, Mochly-Rosen D. A novel Drp1 inhibitor diminishes aberrant mitochondrial fission and neurotoxicity. J Cell Sci. 2012-12-13 [PMID: 23239023]
• Mallozzi C, D'Amore C, Camerini S et al. Phosphorylation and nitration of tyrosine residues affect functional properties of Synaptophysin and Dynamin I, two proteins involved in exo-endocytosis of synaptic vesicles. Biochim Biophys Acta. 2012-01-01 [PMID: 23103755]
• Gonzalez-Flores JN, Gupta N, DeMong LW, Copeland PR. The selenocysteine-specific elongation factor contains a novel and multi-functional domain. J Biol Chem. 2012-01-01 [PMID: 22992746]
• Manczak M, Reddy PH. Abnormal interaction of VDAC1 with amyloid beta and phosphorylated tau causes mitochondrial dysfunction in Alzheimer's disease Hum Mol Genet 2012-09-17 [PMID: 22926141] (Mouse)
• Gopalakrishnan J, Chim YC, Ha A et al. Tubulin nucleotide status controls Sas-4-dependent pericentriolar material recruitment. Nat Cell Biol 2012-01-01 [PMID: 22729084]
• Han JM, Jeong SJ, Park MC et al. Leucyl-tRNA Synthetase Is an Intracellular Leucine Sensor for the mTORC1-Signaling Pathway. Cell 2012-01-01 [PMID: 22424946]
• Xiong Y, Yuan C, Chen R et al. ArfGAP1 Is a GTPase Activating Protein for LRRK2: Reciprocal Regulation of ArfGAP1 by LRRK2. J Neurosci 2012-01-01 [PMID: 22423108]
• Stafa K, Trancikova A, Webber PJ et al. GTPase Activity and Neuronal Toxicity of Parkinson's Disease-Associated LRRK2 Is Regulated by ArfGAP1. PLoS Genet. 2012-01-01 [PMID: 22363216]
• Wang Z, Jiang H, Chen S et al. The Mitochondrial Phosphatase PGAM5 Functions at the Convergence Point of Multiple Necrotic Death Pathways. Cell 2012-01-01 [PMID: 22265414]
• Shirendeb UP, Calkins M, Manczak M et al. Mutant huntingtin’s interaction with mitochondrial protein Drp1 impairs mitochondrial biogenesis and causes defective axonal transport and synaptic degeneration in Huntington’s disease. Human Molecular Genetics. 2011-10-13 [PMID: 21997870]
• Pungaliya PP, Bai Y, Lipinski K et al. Identification and Characterization of a Leucine-Rich Repeat Kinase 2 (LRRK2) Consensus Phosphorylation Motif. PLoS One 2010-01-01 [PMID: 21060682]
• Xiong Y, Coombes CE, Kilaru A et al. GTPase activity plays a key role in the pathobiology of LRRK2. PLoS Genet. 2010-01-01 [PMID: 20386743]
• Wang Y, Liang D, Wang S et al. Role of the G-protein and tyrosine kinase-Rho/ROK pathways in 15-hydroxyeicosatetraenoic acid induced pulmonary vasoconstriction in hypoxic rats. J Biochem 2010-01-01 [PMID: 20139061]
• Wan T, Liu T, Zhang H et al. AIP1 Functions as Arf6-GAP to Negatively Regulate TLR4 Signaling. JBC 2010-01-01 [PMID: 19948740]
• Richter K, Brar S, Ray M et al. Speckled-like Pattern in the Germinal Center (SLIP-GC), a Nuclear GTPase Expressed in Activation-induced Deaminase-expressing Lymphomas and Germinal Center B Cells. JBC 2009-01-01 [PMID: 19734146]
• Tu C, Zhou X, Tropea JE et al. Structure of ERA in complex with the 3' end of 16S rRNA: Implications for ribosome biogenesis. PNAS 2009-01-01 [PMID: 19706445]
• Marino SF. High-level production and characterization of a G-protein coupled receptor signaling complex. FEBS J. 2009-01-01 [PMID: 19645726]
• Villarroya M, Prado S, Esteve JM et al. Characterization of Human GTPBP3, a GTP-Binding Protein Involved in Mitochondrial tRNA Modification. Mol Cell Biol. 2008-01-01 [PMID: 18852288]
• Garcia-Regalado A, Guzman-Hernandez ML, Ramirez-Rangel I et al. G Protein-Coupled Receptor-Promoted Trafficking of G beta-1-gamma-2 Leads to AKT Activation at Endosomes via a Mechanism Mediated by G?1?2-Rab11a Interaction. Mol Biol Cell. 2008-01-01 [PMID: 18701709] (EnzAct, E. coli)
• Moertel L, Gobert GN, McManus DP. Comparative real-time PCR and enzyme analysis of selected gender-associated molecules in Schistosoma japonicum. Parasitology 2008-01-01 [PMID: 18294425]

FAQs for GTPase Kit (602-0120). (Showing 1 - 7 of 7 FAQs).

1. May we have the protocol for 602-0120? The GTPase assay kit protocol seems insufficient.
   • Here is the corresponding full protocol for your customer for this kit. I hope they find this information useful.
2. 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.
3. 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.
4. 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.
5. 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.
6. This is regarding the GTPase assay kit catalog No: 602-0120. I want to know what is the equipment one needs to take the reading as kit contents are pipetted out in a 96-well plate. Does this require a fluorescence plate reader or will it be okay to take reading on the spectrophotometer if a cuvette is used.
   • It is possible to carry out the detection step using cuvettes, but sufficient volume for the cuvette must be used, and note that the PiColorLock solution may damage the cuvette.
7. At which temperature does the assay have to be performed? 
   • The assay can be carried out at room temperature, as the kit contains proprietary buffers which reduce background hydrolysis (the assay is less temperature sensitive because of this). Please note that it is important for all reagents to be warmed up to room temperature before the assay is carried out.