33p atp

Manufactured by PerkinElmer

Sourced in United States

33P ATP is a radioactive nucleotide used in various biochemical and molecular biology applications. It is a phosphorus-33 labeled adenosine triphosphate (ATP) molecule, which serves as a substrate or labeling reagent in experiments involving enzyme activity, nucleic acid synthesis, and other processes requiring ATP.

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Lab products found in correlation

Adenosine triphosphate (atp), by Merck Group (10 mentions) P81 ion exchange filter paper, by Cytiva (7 mentions) Labcyte echo550, by Beckman Coulter (1 mentions) T4 dna polynucleotide kinase, by New England Biolabs (1 mentions) 33p pi, by PerkinElmer (1 mentions) 3h leucine, by PerkinElmer (1 mentions) Bradford reagent, by Bio-Rad (1 mentions) Kinase glo plus luminescence kinase assay kit, by Promega (1 mentions) Adp glo, by Promega (1 mentions) Kinase buffer, by Cell Signaling Technology (1 mentions) Flag m2 agarose beads, by Merck Group (1 mentions) Protease inhibitor, by Roche (1 mentions)

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[γ-33P]ATP (49 citations)

18 protocols using 33p atp

In Vitro Protein Kinase Profiling

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In vitro profiling of a 35 selected member protein kinase panel (see ESI‡) was performed at Reaction Biology Corporation using the “HotSpot” assay platform. Briefly, specific kinase/substrate pairs along with required cofactors^{78 (link)} were prepared in reaction buffer; 20 mM Hepes pH 7.5, 10 mM MgCl₂, 1 mM EGTA, 0.02% Brij35, 0.02 mg mL^–1 BSA, 0.1 mM Na₃VO₄, 2 mM DTT, 1% DMSO. Compounds were delivered into the reaction, followed ∼20 min later by addition of a mixture of ATP (Sigma) and ³³P-ATP (Perkin Elmer) to a final concentration 10 μM. Reactions were carried out at 25 °C for 120 min, followed by spotting of the reactions onto P81 ion exchange filter paper (Whatman). Unbound phosphate was removed by extensive washing of filters in 0.75% phosphoric acid. After subtraction of background derived from control reactions containing inactive enzyme, kinase activity data were expressed as the percent remaining kinase activity in test samples compared to vehicle (dimethyl sulfoxide) reactions. IC₅₀ values and curve fits were obtained using Prism (GraphPad Software).

Fernández-Gallardo J., Elie B.T., Sadhukha T., Prabha S., Sanaú M., Rotenberg S.A., Ramos J.W, & Contel M. (2015). Heterometallic titanium–gold complexes inhibit renal cancer cells in vitro and in vivo †This paper is dedicated to Prof. Roberto Sánchez-Delgado, great mentor and excellent friend, on the occasion of his 65th birthday. ‡Electronic supplementary information (ESI) available: Stability studies of the new compounds by NMR, UV-vis spectroscopy and MS spectrometry, crystallographic data for compound 6, DFT calculations for compounds 4–7, IC50 values in human renal cells at both 24 and 72 h, details on migration studies, TrxR inhibition studies for 3, 5 and AF at different times, inhibition studies of compound 5 against a panel of 35 protein kinases, effects of AF on MAPKAPK-3 in Caki-1 cells, effects of compound 3 in Caki-1 mouse xenografts. CCDC 1400886. For ESI and crystallographic data in CIF or other electronic format see DOI: 10.1039/c5sc01753j Click here for additional data file. Click here for additional data file.. Chemical Science, 6(9), 5269-5283.

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In Vitro Kinase Inhibition Assay

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In vitro assay of the 18 kinases was performed at Reaction Biology Corporation. Kinase and substrate pairs, along with the required cofactors, were prepared in base reaction buffer: 20 mM Hepes pH 7.5, 10 mM MgCl₂, 1 mM EGTA, 0.02% Brij35, 0.02 mg/mL BSA, 0.1 mM Na₃VO₄, 2 mM DTT, 1% DMSO. Compounds in 100% DMSO were delivered into the kinase reaction mixture by Acoustic technology (Labcyte^® Echo550; nanoliter range), followed 20 min later by the addition of a mixture of ³³P-ATP (PerkinElmer, Shelton, CT, USA; 10 µCi/µL) to a concentration of 10 µM. Reactions were carried out at 25 °C for 120 min, followed by spotting of the reactions onto Whatman^® P81 ion exchange filter paper. Unbound phosphate was removed by extensive washing of filters in 0.75% phosphoric acid. After the subtraction of background derived from the control reactions containing inactive enzyme, kinase activity data were expressed as the percentages of remaining kinase activity in test samples compared to vehicle (DMSO) reactions.

Kwon Y.M., Kim S.H., Jung Y.S, & Kwak J.H. (2021). Synthesis and Biological Evaluation of (S)-2-(Substituted arylmethyl)-1-oxo-1,2,3,4-tetrahydropyrazino[1,2-a]indole-3-carboxamide Analogs and Their Synergistic Effect against PTEN-Deficient MDA-MB-468 Cells. Pharmaceuticals, 14(10), 974.

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NF-κB p65 Binding Assay Protocol

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PCR primers were labeled with [-33P]-ATP (Perkin Elmer, Waltham, MA, USA) using T4 DNA polynucleotide kinase (New England Biolabs, Ipswich, MA, USA). Subsequently, DNA probes were created by labeling the forward primers and unlabeling the reverse primers via PCR. Two micrograms of nuclear protein were incubated with a biotin-labeled NF-κB p65 binding-site DNA probe (5′-AGTTGAGGGGACTTTCCCAGGC-3′; Sigma Genosys) in buffer for 30 minutes on ice. Gels were placed on Whatman paper, vacuum-dried, and exposed to a phosphoscreen for 12 hours. Subsequently, the screen was scanned using a storm 860 PhosphorImager, and the data were analyzed using ImageQuant software (Molecular Dynamics/GE Healthcare, Amersham, UK).

Wang Y., Guo X., Jiao G., Luo L., Zhou L., Zhang J, & Wang B. (2019). Splenectomy Promotes Macrophage Polarization in a Mouse Model of Concanavalin A- (ConA-) Induced Liver Fibrosis. BioMed Research International, 2019, 5756189.

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Microautoradiography of Microbial Uptake

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For microautoradiography, triplicate samples (20 ml for GKS, 10 ml for PIB) and one formaldehyde‐killed blank (2% final concentration) were incubated in the dark at in situ temperature with one of the following three substrates purchased from Perkin Elmer: ³³P‐Pi (specific activity 155.8 Ci mg⁻¹; final concentration 50 pM), ³³P‐ATP (specific activity 3000 Ci mmol⁻¹; final concentration 200 pM) and ³H‐leucine (specific activity 56 Ci mmol⁻¹; final concentration 20 nM). Incubations lasted 2–3 h in the case of ³³P‐Pi and ³³P‐ATP, and 1–2 h for ³H‐leucine and were stopped by adding formaldehyde (2% final concentration). Samples were kept overnight at 4°C and filtered on the next day onto 0.22 µm polycarbonate white filters (Millipore GTTP). Filters were rinsed with 5–10 ml of 0.22 µm filtered MQ‐water and stored frozen until further processing.

Rofner C., Sommaruga R, & Teresa Pérez M. (2016). Phosphate and ATP uptake by lake bacteria: does taxonomical identity matter?. Environmental Microbiology, 18(12), 4782-4793.

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Kinase Activity Profiling and Inhibition Assay

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In vitro profiling
of 34 selected member kinase panel was performed at Reaction
Biology Corporation using the “HotSpot” assay platform.
Briefly, specific kinase/substrate pairs along with required cofactors^{70 (link)} were prepared in reaction buffer: 20 mM Hepes
pH 7.5, 10 mM MgCl₂, 1 mM EGTA (ethylene glycol tetracetic
acid), 0.02% Brij35, 0.02 mg/mL BSA (bovine serum albumin), 0.1 mM
Na₃VO₄, 2 mM DTT (dithioethreitol), 1% DMSO.
Compounds were delivered into the reaction, followed ∼20 min
later by addition of a mixture of ATP (Sigma) and ³³P-ATP
(PerkinElmer) to a final concentration 10 μM. Reactions were
carried out at 25 °C for 120 min, followed by spotting of the
reactions onto P81 ion exchange filter paper (Whatman). Unbound phosphate
was removed by extensive washing of filters in 0.75% phosphoric acid.
After subtraction of background derived from control reactions containing
inactive enzyme, kinase activity data were expressed as the percent
remaining kinase activity in test samples compared to vehicle (dimethyl
sulfoxide) reactions. IC₅₀ values and curve fits were obtained
using Prism (GraphPad Software, La Jolla, CA, USA).

Fernández-Gallardo J., Elie B.T., Sulzmaier F.J., Sanaú M., Ramos J.W, & Contel M. (2014). Organometallic Titanocene–Gold Compounds as Potential Chemotherapeutics in Renal Cancer. Study of their Protein Kinase Inhibitory Properties. Organometallics, 33(22), 6669-6681.

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In vitro Kinase Inhibitor Profiling

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In vitro profiling of the 9 structurally validated type II inhibitors was carried out against a large kinase panel comprising of 350 recombinant human protein kinases using the Reaction Biology Corporation “HotSpot” miniaturized kinase assay platform.
All inhibitors were tested at a concentration of 0.5 μM in the presence of 10 μM ATP. Briefly, specific kinase/substrate pairs along with required cofactors were prepared in Base reaction buffer; 20 mM Hepes pH 7.5, 10 mM MgCl2, 1 mM EGTA, 0.02% Brij35, 0.02 mg/ml BSA, 0.1 mM Na3VO4, 2 mM DTT, 1% DMSO. Compounds were delivered into the reaction mixture, followed ~20 min later by addition of a mixture of ATP (Sigma) and ³³P ATP (PerkinElmer) to a final concentration of 10 μM. Reactions were carried out at 25 °C for 120 min, followed by spotting of the reactions onto P81 ion exchange filter paper (Whatman). Unbound phosphate was removed by extensive washing of filters in 0.75% phosphoric acid. After subtraction of background derived from control reactions containing inactive enzyme, kinase activity data were expressed as the percent remaining kinase activity in test samples compared to vehicle (dimethyl sulfoxide) reactions^{29 (link)}.

Vijayan R.S., He P., Modi V., Duong-Ly K.C., Ma H., Peterson J.R., Dunbrack RL J.r, & Levy R.M. (2014). Conformational Analysis of the DFG-Out Kinase Motif and Biochemical Profiling of Structurally Validated Type II Inhibitors. Journal of Medicinal Chemistry, 58(1), 466-479.

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EGFR Tyrosine Kinase Inhibition Assay

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Compounds 1-5 were dissolved in DMSO and tested at a single concentration of 100 µM. They were then added to reaction plates containing the EGFR tyrosine kinase in assay buffer [20 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), pH 7.5, 10 mM MgCl₂, 1 mM ethylene glycol tetraacetic acid (EGTA), 0.02 % Brij35, 0.02 mg/mL bovine serum albumin (BSA), 0.1 mM Na₃VO₄, 2 mM dithiothreitol (DTT), 1 % DMSO]. Reactions were initiated by addition of a mixture of ATP (Sigma, St. Louis, MO) and 33P ATP (Perkin Elmer, Waltham MA) to a final concentration of 10 µM. Reactions were carried out at room temperature for 120 min, followed by spotting of the reactions onto P81 ion exchange filter paper (Whatman Inc., Piscataway, NJ). Unbound phosphate was removed by extensive washing of filters in 0.75 % phosphoric acid (Ma et al., 2008[21 (link)]). Kinase activity data was reported as the percent remaining enzyme activity after subtraction of enzyme inhibitory activity of DMSO control reactions as background. Results are presented as percentage enzyme inhibition and compared to staurosporine as a reference EGFR-TK inhibitor (Table 2(Tab. 2)).

Gabr M.T., El-Gohary N.S., El-Bendary E.R, & El-Kerdawy M.M. (2014). EGFR tyrosine kinase targeted compounds: in vitro antitumor activity and molecular modeling studies of new benzothiazole and pyrimido[2,1-b]benzothiazole derivatives. EXCLI Journal, 13, 573-585.

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Kinase Inhibitory Activity Assay

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“The kinase inhibitory activity of the synthesized compounds was determined using the Kinexus compound profiling service, Canada. Compounds were tested at 50 nM concentration. The kinase used was cloned, expressed and purified using proprietary methods. Quality control testing is routinely performed to ensure compliance to acceptable standards. ³³P-ATP was purchased from PerkinElmer. All other materials were of standard laboratory grade”.

Amr A.E., Ibrahimd A.A., El-Shehry M.F., Hosni H.M., Fayed A.A, & Elsayed E.A. (2019). In Vitro and In Vivo Anti-Breast Cancer Activities of Some Newly Synthesized 5-(thiophen-2-yl)thieno-[2,3-d]pyrimidin-4-one Candidates. Molecules, 24(12), 2255.

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Kinase Profiling of Type II Inhibitors

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In vitro profiling of the nine structurally
validated type II inhibitors was carried out against a large kinase
panel comprising 350 recombinant human protein kinases using the Reaction
Biology Corporation “HotSpot” miniaturized kinase assay
platform.
All inhibitors were tested at a concentration of 0.5
μM in the presence of 10 μM ATP. Briefly, specific kinase/substrate
pairs along with required cofactors were prepared in base reaction
buffer: 20 mM Hepes, pH 7.5, 10 mM MgCl₂, 1 mM EGTA, 0.02%
Brij35, 0.02 mg/mL BSA, 0.1 mM Na₃VO₄, 2 mM
DTT, 1% DMSO. Compounds were delivered into the reaction mixture,
followed ∼20 min later by addition of a mixture of ATP (Sigma)
and ³³P ATP (PerkinElmer) to a final concentration of 10
μM. Reactions were carried out at 25 °C for 120 min, followed
by spotting of the reactions onto P81 ion exchange filter paper (Whatman).
Unbound phosphate was removed by extensive washing of filters in 0.75%
phosphoric acid. After subtraction of background derived from control
reactions containing inactive enzyme, kinase activity data were expressed
as the percent remaining kinase activity in test samples compared
to vehicle (dimethyl sulfoxide) reactions (Figure 10).^{29 (link)}

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Quantifying Sphingolipid Enzyme Activities

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The activity of nSMase2 was measured using radiolabeled [methyl-¹⁴C]choline-sphingomyelin (Perkin-Elmer), and SK1 activity was determined in HMEC-1 lysed in ice-cold lysis buffer, after incubation with [³³P]ATP (Perkin-Elmer), as reported [35 (link), 36 (link)]. The [³³P]-labeled-S1P was extracted, isolated by TLC, and counted by liquid scintillation.
Protein concentration was determined using the Bradford reagent (Bio-Rad).

Camaré C., Vanucci-Bacqué C., Augé N., Pucelle M., Bernis C., Swiader A., Baltas M., Bedos-Belval F., Salvayre R, & Nègre-Salvayre A. (2017). 4-Hydroxynonenal Contributes to Angiogenesis through a Redox-Dependent Sphingolipid Pathway: Prevention by Hydralazine Derivatives. Oxidative Medicine and Cellular Longevity, 2017, 9172741.

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