Malachite Green Phosphate Assay Kit

Produktnummer ABIN1000334

Produktdetails

Target: Malachite Green

Detektionsbereich: 0.4-50 μM

Untere Nachweisgrenze: 0.4 μM

Sensitivität: 20 pmol

Produktmerkmale: Reagent very stable. Due to our innovative formulation, no precipitation of reagent occurs. Therefore no filtration of reagent is needed prior to assays, as is often required with other commercial kits. High sensitivity and wide detection range: detection of as little of 1.6 pmoles of phosphate and useful range between 0.02 µM and 40 µM phosphate.
Fast and convenient: homogeneous mix-and-measure assay allows quantitation of free phosphate within 20 minutes.
Compatible with routine laboratory and HTS formats: assays can be performed in tubes, cuvettes or microplates, on spectrophotometers and plate readers.
Robust and amenable to HTS: Z' factors of 0.7 to 0.9 are observed in 96- well and 384-well plates. Can be readily automated on HTS liquid handling systems.

Bestandteile: Reagent A: 50 mL. Reagent B: 1 mL. Standard: 1mL 1 mM phosphate.

Anwendungsinformationen

Applikationshinweise: Phosphatase Assays: liberation of phosphate from peptide, protein or small molecule substrate. Lipase Assays: liberation of phosphate from phospholipids Nucleoside Triphosphatase Assays: liberation of phosphate from nucleoside triphosphates (ATP, GTP, TTP, CTP etc). Quantitation of Phosphate in phospholipids, proteins and DNAs, etc.
Drug Discovery: high-throughput screen for phosphatase inhibitors.

Kommentare:

Incubation time: The chromogenic reaction is completed within 30 min at room temperature. Read OD values at 30 min. Precipitation may occur at high concentrations of phosphate (>100 μM), or in the presence of high concentrations of e.g. proteins and metals. If precipitation occurs, perform a series dilution of sample in H2O, run the assay and determine the dilution factor from wells with no precipitation. Repeat assays using diluted samples. Enzyme reaction buffer. Because any exogenous free phosphate would interfere with the assay, it is important to ensure that the protein preparation, the reaction buffer and lab wares employed in the assay should not contain free phosphate. This can be conveniently checked by adding the Working Reagent to the buffer and measuring the color formation. Liquid disposal. The assay mixture contains 0.4 M sulfuric acid. It is recommended that the waste liquid be neutralized with equal volume of 1 N NaOH prior to disposal.

Protokoll: 1. Preparation of phosphate standards. Prepare a Premix solution containing 40 µMphosphate by pipetting 40 µL 1 mM phosphate standard to 960 µL distilled water or enzyme reaction buffer. Number the tubes. Dilute standards as shown in the following Table. Pipette 80 µL standard in duplicate into wells of a clear-bottom 96-well plate. Add blank controls containing water or reaction buffer only.
2. Transfer 80 µL test samples into separate wells of the plate. Note: in the case of enzyme reactions, the reaction may be terminated by adding a specific inhibitor, or can be stopped directly by the addition of the Working Reagent. Dilution of reaction mixture may be necessary prior to the assay (see General Considerations). For ATPase or GTPase assays, the ATP or GTP concentration should be lower than 0.25 mM. If the reaction mixture contains > 0.25mM ATP or GTP, dilute samples in distilled water. For example, if the ATPase reaction contained 1 mM ATP, at the end of reaction dilute reaction mixture 4-fold in water prior to the assay.
3. Add 20 µL of Working Reagent to each well. Mix gently by tapping the plate.
4. Incubate for 30 min at room temperature for color development.
5. Measure absorbance at 600 nm - 660nm (620 nm) on a plate reader. For assays in 384-well plates, the procedures are the same, except that the volume of the standard and sample solution should be 40 µL and that of the Working Reagent should be 10 µL.

Aufbereitung der Reagenzien:

Each assay requires 20 µL Working Reagent. Prepare enough Working Reagent by mixing 100 vol of Reagent A and 1 vol of Reagent B (e.g. 5 mL Reagent A and 50 µL Reagent B). Working Reagent is stable for at least 1 day at room temperature. Important: The reagent must be brought to room temperature before use. Before each assay, it is important to check that all enzyme preparations and assay buffers do not contain free phosphate. This can be conveniently done by adding 20 µL of the Working Reagent to 80 µL sample solution. The blank OD values at 620 nm should be lower than 0.2. If the OD readings are higher than 0.2, check water phosphate level. Double distilled water usually have OD readings lower than 0.1. Lab detergents may contain high levels of phosphate. Make sure that lab wares are free from contaminating phosphate after thorough washes.

Beschränkungen: Nur für Forschungszwecke einsetzbar

Handhabung

Lagerung: 4 °C

Referenzen

Cha, Kim, Kim, Kong: "Integrative design of a poly(ethylene glycol)-poly(propylene glycol)-alginate hydrogel to control three dimensional biomineralization." in: Biomaterials, Vol. 32, Issue 11, pp. 2695-703, (2011) (PubMed).

Reigan, Siegel, Guo, Ross: "A mechanistic and structural analysis of the inhibition of the 90-kDa heat shock protein by the benzoquinone and hydroquinone ansamycins." in: Molecular pharmacology, Vol. 79, Issue 5, pp. 823-32, (2011) (PubMed).

Sinkunas, Gasiunas, Fremaux, Barrangou, Horvath, Siksnys: "Cas3 is a single-stranded DNA nuclease and ATP-dependent helicase in the CRISPR/Cas immune system." in: The EMBO journal, Vol. 30, Issue 7, pp. 1335-42, (2011) (PubMed).

Lin, Wilson: "Escherichia coli thioredoxin-like protein YbbN contains an atypical tetratricopeptide repeat motif and is a negative regulator of GroEL." in: The Journal of biological chemistry, Vol. 286, Issue 22, pp. 19459-69, (2011) (PubMed).

Aldana-Masangkay, Rodriguez-Gonzalez, Lin, Ikeda, Hsieh, Kim, Lomenick, Okemoto, Landaw, Wang, Mazitschek, Bradner, Sakamoto: "Tubacin suppresses proliferation and induces apoptosis of acute lymphoblastic leukemia cells." in: Leukemia & lymphoma, Vol. 52, Issue 8, pp. 1544-55, (2011) (PubMed).

Demidenko, Lee, Powers, Nibert: "Effects of viscogens on RNA transcription inside reovirus particles." in: The Journal of biological chemistry, Vol. 286, Issue 34, pp. 29521-30, (2011) (PubMed).

Li, Uitto: "The mineralization phenotype in Abcc6 ( -/- ) mice is affected by Ggcx gene deficiency and genetic background--a model for pseudoxanthoma elasticum." in: Journal of molecular medicine (Berlin, Germany), Vol. 88, Issue 2, pp. 173-81, (2010) (PubMed).

Hsieh, Lin, Lai, Lin, Hsieh, Shih: "Direct MinE-membrane interaction contributes to the proper localization of MinDE in E. coli." in: Molecular microbiology, Vol. 75, Issue 2, pp. 499-512, (2010) (PubMed).

Yoshida, Hishiyama, Miyazaki, Watanabe, Kanbe, Yamada, Matsuzaki, Miyashita, Kitanaka, Miyata: "Analysis of inhibition of topoisomerase IIalpha and cancer cell proliferation by ingenolEZ." in: Cancer science, Vol. 101, Issue 2, pp. 374-8, (2010) (PubMed).

Guilfoyle, Maher, Rapp, Clarke, Harrop, Jormakka: "Structural basis of GDP release and gating in G protein coupled Fe2+ transport." in: The EMBO journal, Vol. 28, Issue 17, pp. 2677-85, (2009) (PubMed).

Barisic, Strozyk, Peters, Walczak, Kulms: "Identification of PP2A as a crucial regulator of the NF-kappaB feedback loop: its inhibition by UVB turns NF-kappaB into a pro-apoptotic factor." in: Cell death and differentiation, Vol. 15, Issue 11, pp. 1681-90, (2008) (PubMed).

Ramamurthi, Losick: "ATP-driven self-assembly of a morphogenetic protein in Bacillus subtilis." in: Molecular cell, Vol. 31, Issue 3, pp. 406-14, (2008) (PubMed).

Koroleva, Makharashvili, Courcelle, Courcelle, Korolev: "Structural conservation of RecF and Rad50: implications for DNA recognition and RecF function." in: The EMBO journal, Vol. 26, Issue 3, pp. 867-77, (2007) (PubMed).

Lu, Yadav, Shah, Liu, Tian, Pukszta, Villaluna, Kutejová, Newlon, Santos, Suzuki: "Roles for the human ATP-dependent Lon protease in mitochondrial DNA maintenance." in: The Journal of biological chemistry, Vol. 282, Issue 24, pp. 17363-74, (2007) (PubMed).

Anand, Zheng, Bianco, Leuba, Khan: "DNA helicase activity of PcrA is not required for the displacement of RecA protein from DNA or inhibition of RecA-mediated strand exchange." in: Journal of bacteriology, Vol. 189, Issue 12, pp. 4502-9, (2007) (PubMed).

Adkins, Williams, Linger, Tyler: "Chromatin disassembly from the PHO5 promoter is essential for the recruitment of the general transcription machinery and coactivators." in: Molecular and cellular biology, Vol. 27, Issue 18, pp. 6372-82, (2007) (PubMed).

Guérette, Khan, Savard, Vincent: "Molecular evolution of type VI intermediate filament proteins." in: BMC evolutionary biology, Vol. 7, pp. 164, (2007) (PubMed).

Saran, Held, Burke: "Multiple-turnover thio-ATP hydrolase and phospho-enzyme intermediate formation activities catalyzed by an RNA enzyme." in: Nucleic acids research, Vol. 34, Issue 11, pp. 3201-8, (2006) (PubMed).

Guo, Reigan, Siegel, Zirrolli, Gustafson, Ross: "The bioreduction of a series of benzoquinone ansamycins by NAD(P)H:quinone oxidoreductase 1 to more potent heat shock protein 90 inhibitors, the hydroquinone ansamycins." in: Molecular pharmacology, Vol. 70, Issue 4, pp. 1194-203, (2006) (PubMed).

Chao, Xing, Chen, Xu, Lin, Li, Jeffrey, Stock, Shi: "Structure and mechanism of the phosphotyrosyl phosphatase activator." in: Molecular cell, Vol. 23, Issue 4, pp. 535-46, (2006) (PubMed).

Antigendetails

Target: Malachite Green

Andere Bezeichnung: Malachite Green Phosphate

Hintergrund: High-throµghput phosphate assay using malachite green method at 620nm.

The Malachite Green Phosphate Assay Kit is based on quantification of the green complex formed between Malachite Green, molybdate and free orthophosphate. The rapid color formation from the reaction can be conveniently measured on a spectrophotometer (600 - 660 nm) or on a plate reader. The non-radioactive colorimetric assay kits have been optimized to offer superior sensitivity and prolonged shelf life. The assay is simple and fast, involving a single addition step for phosphate determination. Assays can be executed in tubes, cuvettes or multi-well plates. The assays can be conveniently performed in 96- and 384-well plates for high-throughput screening of enzyme inhibitors.