CD8+ T cells and 293T cells were lysed in IP lysis buffer (Pierce). For immunoprecipitation experiments Flag-BACH2/BACH2(S520A) was immunoprecipitated following overnight incubation with M-2 anti-Flag monoclonal antibodies (Sigma) using protein A/G magnetic beads (Dynal). After several washes, immunoprecipitated proteins were released from beads using competitive elution with Flag peptide according to manufacturer’s instructions (Sigma) or by boiling in 1x Laemmli buffer. Proteins similarly immunoprecipitated from 293T cells transfected with WT or BACH2(S520A) were subjected to kinase assays while bound to beads and in the presence of ATP (Sigma) and kinase reaction buffer according to manufacturer’s instructions (Cell Signaling).
Kinase reaction buffer
Manufactured by Cell Signaling Technology
Sourced in United States
Kinase reaction buffer is a solution designed to provide the optimal conditions for kinase enzyme-mediated phosphorylation reactions. The buffer maintains the appropriate pH, ionic strength, and cofactor concentrations necessary for kinase activity.
Automatically generated - may contain errors
Lab products found in correlation
Adenosine triphosphate (atp), by Merck Group (3 mentions)
M 2 anti flag monoclonal antibodies, by Merck Group (2 mentions)
Protein a g magnetic bead, by Thermo Fisher Scientific (2 mentions)
Flag peptide, by Merck Group (2 mentions)
Adenosine triphosphate (atp), by Cell Signaling Technology (2 mentions)
T β cat, by Cell Signaling Technology (1 mentions)
Horseradish peroxidase conjugated goat anti mouse igg, by Cell Signaling Technology (1 mentions)
Okadaic acid, by Cell Signaling Technology (1 mentions)
9 protocols using kinase reaction buffer
1
Immunoprecipitation and Kinase Assay
2
ASK1 Kinase Activity Assay
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In vitro kinase assays were performed at 30°C for 2.5 h in the kinase reaction buffer (Cell Signaling Technology), by using ASK1 immunoprecipitate and bacterially purified His-EB1 as described previously [18 (link)].
3
Cell-free assay for GSK-3β activity
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Cell-free GSK-3β activity assays were performed as previously described [34] (link). In brief, after IP, the HA-resin was washed twice with Tris-buffered saline with 0.1% Tween, followed by one wash with kinase reaction buffer (Cell Signaling Technology). Next, immunoprecipitated GSK3β (WT and mutant) was resuspended in 50 µL kinase reaction buffer in the presence of 1 μg β-catenin (β-Cat, Sigma-Aldrich) as substrate and 0.4 mM ATP (Cell signaling technology). To test the inhibitory effect of electrophiles, 4-HNE was added to the reaction system at the indicated concentrations for 1 h prior to initiation of the activity assay. The kinase reaction was carried out at 37 °C for 30 min under gentle agitation, and terminated by adding 4× reducing sample buffer (Bio-Rad), and denatured by heating at 100 °C for 5 min. The levels of phospho-β-catenin (p-β-Cat, S33/37/T41, Cell Signaling Technology) and total β-catenin (t-β-Cat, Cell Signaling Technology) were analyzed using Western blotting, and the ratios of p-β-Cat/t-β-Cat were calculated to represent GSK3β activity.
4
ASK1 Kinase Phosphorylation of EB1
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ASK1 and ASK1KD were transfected into 293 T cells and immunoprecipitated with anti-ASK1 antibody. Kinase assays were performed at 30 °C for 2.5 h in the kinase reaction buffer (Cell Signaling Technology, Danvers, MA, USA), by using ASK1 or ASK1KD immunoprecipitate and bacterially purified GST-EB1 or His-EB1. To examine EB1 dephosphorylation, GST-EB1 was pulled down from the above reaction mixture with glutathione-coated agarose beads, incubated with λPPase in the dephosphorylation buffer (Cell Signaling Technology) at 35 °C for 2 h and then examined by immunoblotting.
5
Immunoprecipitation and Kinase Assay
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CD8+ T cells and 293T cells were lysed in IP lysis buffer (Pierce). For immunoprecipitation experiments Flag-BACH2/BACH2(S520A) was immunoprecipitated following overnight incubation with M-2 anti-Flag monoclonal antibodies (Sigma) using protein A/G magnetic beads (Dynal). After several washes, immunoprecipitated proteins were released from beads using competitive elution with Flag peptide according to manufacturer’s instructions (Sigma) or by boiling in 1x Laemmli buffer. Proteins similarly immunoprecipitated from 293T cells transfected with WT or BACH2(S520A) were subjected to kinase assays while bound to beads and in the presence of ATP (Sigma) and kinase reaction buffer according to manufacturer’s instructions (Cell Signaling).
6
Kinase Inhibition Assay with Recombinant Proteins
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STAT5 (reference no. SRP5142), Pim-2 (reference no. K3518), and FLT3 571-993 (reference no. F6432) recombinant proteins were from Sigma-Aldrich and were used at a concentration of 200 ng in a final volume of 100 μl. Proteins were mixed in a kinase reaction buffer (Cell Signaling Technology, reference no. 9802) with 0, 1, 2, 5, 10, or 50 nM AC220 and 0 or 1 μM LGB321 and incubated for 1 hour at 37°C. Then, 200 μM ATP (Cell Signaling Technology, reference no. 9804) was added, and the mixture was further incubated for 30 min at 37°C. The reactions were stopped by the addition of boiling Laemmli buffer.
7
Tyrosine Kinase Inhibition by Compound ZW97
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The inhibitory effects of compound ZW97 against various tyrosine kinases
(anaplastic lymphoma kinase [ALK], fibroblast growth factor receptor 1 [FGFR1],
epidermal growth factor receptor [EGFR], tyrosine kinase A [TRKA],
platelet-derived growth factor receptor α [PDGFRα]) were determined using
enzyme-linked immunosorbent assays (ELISAs). Adenosine triphosphate solution
diluted in kinase reaction buffer (Cell Signaling Technology, Beverly, USA) was
added to each well and compound ZW97 diluted in DMSO was added to each reaction
well. DMSO was used as the negative control. The kinase reaction was initiated
by the addition of purified tyrosine kinase proteins (Cell Signaling
Technology®, Danvers, MA, USA). After incubation for 1 h at 37°C, the plate was
washed three times with 1 mM PBS (pH 7.5) and horse radish peroxidase-conjugated
goat anti-mouse IgG (Cell Signaling Technology®) was added. The plate was then
incubated at 37°C for 30 min and washed three times with 1 mM PBS (pH 7.5) at
37°C for 10 min each time. Upon completion, the plate was analysed using a
spectrophotometer (SKILLBIO). The minimum detectable concentration was 1 pg/ml
for the targeted compound. Intra- and interassay coefficients of variation for
all ELISAs were <0.05% and <0.05%, respectively.
(anaplastic lymphoma kinase [ALK], fibroblast growth factor receptor 1 [FGFR1],
epidermal growth factor receptor [EGFR], tyrosine kinase A [TRKA],
platelet-derived growth factor receptor α [PDGFRα]) were determined using
enzyme-linked immunosorbent assays (ELISAs). Adenosine triphosphate solution
diluted in kinase reaction buffer (Cell Signaling Technology, Beverly, USA) was
added to each well and compound ZW97 diluted in DMSO was added to each reaction
well. DMSO was used as the negative control. The kinase reaction was initiated
by the addition of purified tyrosine kinase proteins (Cell Signaling
Technology®, Danvers, MA, USA). After incubation for 1 h at 37°C, the plate was
washed three times with 1 mM PBS (pH 7.5) and horse radish peroxidase-conjugated
goat anti-mouse IgG (Cell Signaling Technology®) was added. The plate was then
incubated at 37°C for 30 min and washed three times with 1 mM PBS (pH 7.5) at
37°C for 10 min each time. Upon completion, the plate was analysed using a
spectrophotometer (SKILLBIO). The minimum detectable concentration was 1 pg/ml
for the targeted compound. Intra- and interassay coefficients of variation for
all ELISAs were <0.05% and <0.05%, respectively.
8
Src-EB1 Kinase Interaction Assay
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GFP-Src immunoprecipitated from HEK293T cells or bacterially purified GST-Src was incubated with bacterially purified His-EB1 at 30°C for 2.5 h in the kinase reaction buffer (Cell Signaling Technology) containing ATP (200 µM). The reaction samples were then subjected to immunoblotting with pTyr or pY247-EB1 antibodies.
9
Tau Kinase Assay in Mouse Cortical Neurons
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Mouse cortical neuronal cells (T4) were lysed in lysis buffer (50 mM Tris-HCl [pH 7.9], 100 mM NaCl, 10 mM MgCl2, 1 mM DTT) containing 0.1% NP-40 and protease inhibitors. Proteins were extracted on ice with periodic vortexing for 30 min, and lysates were cleared by centrifugation at 10,000 × g for 10 min at 4 °C. Ten micrograms of the lysates was incubated with 2 μg of GST-tau protein at 32 °C for 40 min in a kinase reaction buffer (Cell Signaling Technology) containing 10 μM cAMP (Tocris), 5 nM okadaic acid (Cell Signaling Technology) and 1 mM ATP (Sigma). The reaction was stopped by the addition of 1x SDS-sample loading buffer and incubation at 95 °C for 5 min. Protein was separated by SDS-PAGE and immunoblotted as described below.
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