The antibodies to β-actin (5125), poly(ADP-ribose) polymerase (PARP) (9532S), cleaved caspase-3 (9661S), BTK (8547), PLCγ2(55512), ERK1/2 (9107), phospho-PLCγ2 (Tyr1217) (3871), phospho-BTK (Tyr223) (87457), and ERK1/2 pT202/pY204 (4377) were purchased from Cell Signaling Technology. An Annexin V-PE Apoptosis Detection Kit (555763), and PE mouse anti-human CD69 (555531), PE mouse anti-human CD3 (555340), and PE mouse anti-human CD19 (561741) were purchased from BD Bioscience. Dynabeads® Human T-Activator CD3/CD28 (11161D) was purchased from Life Technology. Goat anti-human IgM F(ab)2 (2022) was purchased from SouthernBiotech. An HTRF® KinEASE™-TK Kit (62TK0PEC) was purchased from Cisbio. Fugene HD Transfection Reagent (04709705001) was purchased from Roche. A Pierce™ BCA Protein Assay Kit (23225) was purchased from Thermo Fisher Scientific. Cell-Titer Blue® (G8081) was purchased from Promega. Human wild-type BTK and BTK C481A mutants were amplified by PCR and cloned into the pcDNA3.1 vector. All expression plasmids were verified by DNA sequencing.
Htrf kinease tk kit
Manufactured by PerkinElmer
Sourced in France
The HTRF KinEASE-TK kit is a homogeneous time-resolved fluorescence (HTRF) assay developed by PerkinElmer to measure the kinase activity of tyrosine kinases. The kit provides a reliable and sensitive method for the evaluation of kinase inhibitors and the screening of kinase activities.
Automatically generated - may contain errors
Lab products found in correlation
Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Pe mouse anti human cd19, by BD (1 mentions)
Pe annexin 5 apoptosis detection kit, by BD (1 mentions)
Celltiter blue, by Promega (1 mentions)
Fugene hd transfection reagent, by Roche (1 mentions)
Infinite f500 microplate reader, by Tecan (1 mentions)
Prism 8, by GraphPad (1 mentions)
Cell mitochondrial isolation kit, by Beyotime (1 mentions)
Annexin 5 fitc apoptotic detection kit, by Beyotime (1 mentions)
Cytochrome c, by Wanlei (1 mentions)
Antibodies against akt, by Cell Signaling Technology (1 mentions)
Bcl 2, by Cell Signaling Technology (1 mentions)
Cleaved caspase 3, by Cell Signaling Technology (1 mentions)
Ly294002, by Beyotime (1 mentions)
Dapi staining solution, by Beyotime (1 mentions)
Infinite f200 pro, by Tecan (1 mentions)
Victorx 5 multilabel reader, by PerkinElmer (1 mentions)
6 protocols using htrf kinease tk kit
1
Molecular Profiling of BTK Signaling Pathway
2
Inhibition Assay for HER2 Kinase
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All enzymatic reactions were carried out at 30°C for 40 min. The 50 µl reaction mixture contained 40 mmol/L Tris, 10 mmol/L MgCl2, 0.1 mg/ml BSA, 1 mmol/L DTT, 10 µmol/L ATP, 0.4 ng/µl HER2 kinase, and 100 µmol/L lipid substrate. The compounds were diluted in 10% DMSO and 5 µl of the dilution was added to a 50 µl reaction; the final concentration of DMSO was 1% in all reactions. These assays were undertaken using the HTRF kinEASE TK kit (Cisbio). The fluorescent signals were correlated with the amount of ATP present and were inversely correlated with kinase activity. The IC50 values were calculated using nonlinear regression with normalized dose‐response fit.
3
FAK Kinase Inhibition Assay Protocol
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The FAK kinase assay was performed using the HTRF® KinEASE™-TK kit (Cisbio Bioassays, France) in white 384-well small volume plates with a total working volume of 20 μL. Purified FAK enzyme was purchased from Carna Biosciences (Japan). Compounds were diluted step-by-step from a concentrated stock of 8 mM in 100% DMSO and with serial kinase reaction buffer dilutions. The IC50 measurements were performed in replicates. For each assay, 4 μL of dispensed compounds, 4 μL of mix 1 (ATP + Substrate TK) and 2 μL of kinase (0.111 ng/mL) were added to the assay wells. The assay plates were incubated at 30 °C for 50 min and reactions were terminated by adding 10 μL of mix 2 (Sa-XL665 + TK-Antibody-Cryptate). After a final incubation (60 min at room temperature), HTRF signals were obtained by reading plates using an Infinite® F500 microplate reader (Tecan, Switzerland). The fluorescence was measured at 620 nM (Cryptate) and 665 nM (XL665). A ratio was calculated (665/620) for each well. For IC50 measurements, values were normalised and fitted with Graphpad prism 8.3.0.
4
Isolation and Characterization of Novel Streptomyces Compounds
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Act V (purity > 98%) and Act D (purity > 98%) were obtained from Dr. Xie (Shandong University, Weihai, China), isolated from Streptomyces sp. [33 (link)]. The compounds were dissolved in dimethyl sulfoxide (DMSO). Antibodies against AKT, PI3K, Bcl-2, Bax cleaved caspase-9, and cleaved caspase-3 were purchased from Cell Signalling Technology (CST Inc., Beverly, MA, USA). Antibodies against p-AKT, cytochrome C, and PARP were purchased from WanLei Biotechnology (Shenyang, China). Antibodies against GAPDH and β-Actin were purchased from Beyotime Institute of Biotechnology (Shanghai, China). The Annexin V-FITC apoptotic detection kit, JC-1 (“5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethyl-imidacarbocyanine iodide”) staining kit, LY294002 (PI3K inhibitor), DAPI staining solution, and cell mitochondrial isolation kit were purchased from Beyotime Institute of Biotechnology (Shanghai, China). The HTRFKinEASE-TK kit was purchased from Cisbio (Cisbio Bioassays Co., Codolet, France). The PI3K antibody in the kinase inhibition assay was obtained from Abcam, Inc. (Cambridge, MA, USA). All chemicals used in this study were commercial reagent grade products.
5
Inhibition of MerTK Kinase Activity
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The inhibitory activity of AZD7762 against MerTK was measured and verified with an HTRF assay, an application of the fluorescence resonance energy-transfer (FRET) technique. The HTRF KinEASE-TK kit (Cisbio, Bedford, MA, USA) was used according to the manufacturer’s instructions. To draw and fit the Michaelis–Menten curves, an HTRF assays at various concentrations of ATP were performed. IC50 values were obtained at 35 µM (close to Km value for ATP from in vitro assay) or 1 mM ATP (ATP concentration in cells), respectively. We used 1 ng/µL of purified recombinant MerTK for the kinase assay, and the reaction mixture contained 0.2% DMSO.
Fluorescence signals were measured at two wavelengths for the HTRF assay. The excitation wavelength was 320 nm, and the emission wavelengths were 620 and 665 nm. Fluorescence was detected using an Infinite F200pro with a lag time of 150 µs and integration time of 500 µs (Tecan, Männedorf, Switzerland). Data reduction was carried out following the manufacturer’s guidelines (Ratio = 10,000 × signal 665 nm/signal 620 nm).
Fluorescence signals were measured at two wavelengths for the HTRF assay. The excitation wavelength was 320 nm, and the emission wavelengths were 620 and 665 nm. Fluorescence was detected using an Infinite F200pro with a lag time of 150 µs and integration time of 500 µs (Tecan, Männedorf, Switzerland). Data reduction was carried out following the manufacturer’s guidelines (Ratio = 10,000 × signal 665 nm/signal 620 nm).
6
Trk Kinase Activity Evaluation by HTRF
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A time-resolved fluorescence-based HTRF KinEASE-TK kit (Cisbio, Codolet, France) was used to evaluate TrkA kinase activity. Recombinant proteins containing the TrkA kinase domain were purchased from Invitrogen (Thermo Fisher Scientific, Waltham, MA, USA). The reaction was performed in a 96-well plate with a kinase reaction mixture containing 0.1 μM TK-substrate biotin, 500 μM ATP, and 1 ng of TrkA kinase with a 3-fold serial dilution of the test compound in kinase reaction buffer (50 mM HEPES [pH 7.0], 5 mM MgCl2 1 mM DTT, 0.1 mM orthovanadate, 0.01% bovine serum albumin [BSA], and 0.02% NaN3). After addition of the detection reagents, the TR-FRET signal was measured with a Victor X5 multilabel reader (Perkin Elmer, Waltham, MA, USA) at 615 nm and 665 nm. An equal amount of 1% DMSO was added to each kinase reaction at every dose point. The dose-response curve was fitted by nonlinear regression, and the IC50 was calculated using Prism version 5.01 (GraphPad Software, San Diego, CA, USA).
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