kinase assay buffer, by Cell Signaling Technology - Product details

1

Characterizing IKK2 Complex Activation

Check if the same lab product or an alternative is used in the 5 most similar protocols

WT and NM-cKO BMMS were treated with RANKL for 0, 20 and 40 minutes followed by lysis in RIPA buffer (50 mM Tris (pH 7.4), 150 mM NaCl, 1% NP-40, 1 mM EDTA 50 μg/ml leupeptin, 10 μg/ml aprotinin, 50 μg/ml phenylmethylsulfonyl fluoride (PMSF), 0.2 mM sodium orthovanadate, 100 mM sodium fluorine) for immunoprecipitation. IKK2 complex was immunoprecipitated with NEMO FL-419 (Santa Cruz, Dallas, TX, USA) antibody, washed twice with IP buffer, once with kinase assay buffer (Cell Signaling Technologies Danvers, MA, USA), and incubated for 30 minutes at 30 °C in 30 μl kinase assay buffer with 1 μg GST-IκBα, 2.5 mM MgCl2, and 16 μM ATP. Reaction was terminated with 10 μl 4 X reducing sample buffer. Samples were analyzed by Western blot.

Swarnkar G., Shim K., Nasir A.M., Seehra K., Chen H.P., Mbalaviele G, & Abu-Amer Y. (2016). Myeloid Deletion of Nemo Causes Osteopetrosis in Mice Owing to Upregulation of Transcriptional Repressors. Scientific Reports, 6, 29896.

+ Open protocol

+ Expand

2

RASSF9 Phosphorylation by TAK1 Kinase

Check if the same lab product or an alternative is used in the 5 most similar protocols

Recombinant RASSF9 (2 µg) was incubated with 1 µg of recombinant TAK1 (Novus, H00006885‐P01) in kinase assay buffer (Cell Signaling, #9802) containing 25 mm Tris‐HCl (pH 7.5), 5 mm beta‐glycerophosphate, 2 mm dithiothreitol, 0.1 mm Na₃VO₄, 10 mm MgCl₂, and 200 µm ATP (Cell Signaling, #9804). The incubation was performed for 30 min at 37 °C. The reaction was terminated by adding 1 × Laemmli buffer. The samples were boiled at 100 °C for 5 min and separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS‐PAGE), and then subjected to Coomassie blue staining or transferred onto a polyvinylidene fluoride membrane. Signals for RASSF9, p‐RASSF9, and TAK1 were detected using the respective antibodies.

Shi H., Ju Q., Mao Y., Wang Y., Ding J., Liu X., Tang X, & Sun C. (2021). TAK1 Phosphorylates RASSF9 and Inhibits Esophageal Squamous Tumor Cell Proliferation by Targeting the RAS/MEK/ERK Axis. Advanced Science, 8(5), 2001575.

+ Open protocol

+ Expand

3

In Vitro Kinase Assay for IKKβ

Check if the same lab product or an alternative is used in the 5 most similar protocols

Recombinant active GST-IKKβ or immunoprecipitated Flag-IKKβ was incubated for 30 min at 30°C with kinase assay buffer (Cell Signaling), 200 µM ATP (10 mM stock; Cell Signaling), the specified recombinant GST-tagged proteins, and 1 µL of [γ-³²P]-ATP (10 mCi per milliliter of stock) in a final volume of 30 µL. The reaction was quenched by adding 10 µL of SDS loading dye, and reactions were separated by SDS-PAGE and exposed to autoradiograph film for 15 min to 1 h. For cold kinase assays, all reaction conditions were the same except for the addition of [γ-³²P]-ATP.

Reid M.A., Lowman X.H., Pan M., Tran T.Q., Warmoes M.O., Ishak Gabra M.B., Yang Y., Locasale J.W, & Kong M. (2016). IKKβ promotes metabolic adaptation to glutamine deprivation via phosphorylation and inhibition of PFKFB3. Genes & Development, 30(16), 1837-1851.

+ Open protocol

+ Expand

4

Kinase activity of Akt with USP14

Check if the same lab product or an alternative is used in the 5 most similar protocols

Recombinant USP14 or USP14 mutant protein (1 μg) was incubated with 1 μg active Akt, 0.2 mM ATP, and kinase assay buffer (Cell Signaling) in a total volume of 50 μl for 1 hr at 30°C. The reaction mixtures were subjected to Ub-AMC assay by the addition of 50 μl 2×Ub-AMC buffer. Alternatively, the kinase reaction was stopped by the addition of 50 μl 2×sample buffer, and resolved by SDS-PAGE, followed by blotting with phospho-specific antibodies.

Xu D., Shan B., Lee B.H., Zhu K., Zhang T., Sun H., Liu M., Shi L., Liang W., Qian L., Xiao J., Wang L., Pan L., Finley D, & Yuan J. (2015). Phosphorylation and activation of ubiquitin-specific protease-14 by Akt regulates the ubiquitin-proteasome system. eLife, 4, e10510.

+ Open protocol

+ Expand

5

In Vitro Binding and Kinase Assays

Check if the same lab product or an alternative is used in the 5 most similar protocols

GST-CDK4 was purchased from Abcam, Inc (Cambridge, MA), while GST-CDK4/cyclin D1 was from Signal Chem, Inc (Richmond, Canada). For GST protein, E. coli. harboring GST or GST-CDK4 were grown overnight in room temperature. The recombinant proteins were induced with 1 mM IPTG for 4 hours. GST was purified by glutathione column and diluted to 1 ng/ul with 1X kinase assay buffer provided by the manufacturer (Cell Signaling, Inc, Danvers, MA). This was followed by combining 25 μl GST or GST-CDK4 with 1 ng substrate (GST-Rb) with or without 25 ng of cyclin D1, or HisTAG-MCM8, or HisTAG- ΔMCM8^del261-290 or 2 ng of HisTAG-MCM8CBM in 1xkinase reaction buffer. The solutions were incubated at 37°C for 60 minutes. The reactions were terminated by adding 25 μl of 2N NaOH stop solution to each reaction well. The kinase activities were quantified using the kit and protocols of ADP-Glo™ Kinase Assay from Promega, Inc, Madison, WI.
For in vitro MCM8/cyclin D1/CDK4 binding assay, the following condition was used: 25 ng of each of HisTAG-MCM8, GST-cyclinD1 and GST-CDK4 produced from E.coli. were incubated with 150 mM NaCl, 25 mM MOPS, pH 7.2, 12.5 mM MgCl₂, 12.5 mM β-glycerol phosphate at 37°C for 2 h. The products were then resolved in 6% non-denaturing polyacrylamide gel electrophoresis.

He D.M., Ren B.G., Liu S., Tang L.Z., Cieply K., Tseng G., Yu Y.P, & Luo J.H. (2017). Oncogenic Activity of Amplified Miniature Chromosome Maintenance 8 in Human Malignancies. Oncogene, 36(25), 3629-3639.

+ Open protocol

+ Expand

6

IKK Complex Kinase Assay Protocol

Check if the same lab product or an alternative is used in the 5 most similar protocols

MEFs were lysed in RIPA buffer [20 mM Tris-HCl pH 7.5, 150 mM NaCl, 1 mM EDTA, 1% Triton-X100, 0.5% Na deoxycholate, 0.1% SDS, 1.25 mM NaF, 0.2 mM NaVO₃, 2 mM β-glycerophosphate and complete TM protease inhibitor cocktail (Roche)], each lysate normalized to 500 g total protein, and IKK complex was immunoprecipitated using 1 μg of a sheep polyclonal anti-IKKγ antibody and 15 μl Protein G Dynabeads (Thermo) by incubation at 4 °C for 1 h. Beads were then washed 3 times in RIPA buffer and 1 time in kinase assay buffer (Cell Signaling #9802), and kinase assays were performed in kinase assay buffer plus 1 mM ATP and 2 μg of GST-IκBα 1–54 peptide in a total volume of 20 μl for 30 min at 30 °C. The reaction was terminated with 7.5 μl 4X LDS buffer (Thermo NP0007) plus 1 μl 14.3 M β-mercaptoethanol. Kinase activity was determined by immunoblot analysis using a p-IκBα specific antibody. Antibodies used were: TNFR (Cell Signaling C25C1), NEMO (R&D AF4365, LSBio LS-C346449, or SCBT sc-166398), tubulin (Millipore 05–829), RIPK1 (Cell Signaling 3493), p-IKKα/β (CST 2078S), total IKK (CST: 8943S), (p-IκBα: CST: CS9246), total IκBα (CST: 9242L), Pro-IL-1β (BioLegend B122), GST (BioLegend 640802), A20 (Cell Signaling 5630S), Pro-Caspase8 (Cell Signaling D35G2).

Razani B., Whang M.I., Kim F.S., Nakamura M.C., Sun X., Advincula R., Turnbaugh J.A., Pendse M., Tanbun P., Achacoso P., Turnbaugh P.J., Malynn B.A, & Ma A. (2020). Non-catalytic ubiquitin binding by A20 prevents psoriatic arthritis-like disease and inflammation. Nature immunology, 21(4), 422-433.

+ Open protocol

+ Expand

7

AKT2 Variant Kinase Assay

Check if the same lab product or an alternative is used in the 5 most similar protocols

We isolated V5-AKT2, V5-AKT2.Lys17, V5-AKT2.Thr50, V5-AKT2.Lys208, V5-AKT2.His274, and V5-AKT2.Trp467 variants from lentivirally infected and 5μg/mL blasticidin selected HeLa cell lysate with V5 agarose beads (SIGMA) and incubated with 150ng GST-GSK3β substrate peptide (Cell Signaling Technologies) and 250mM cold ATP in kinase assay buffer (Cell Signaling Technologies) for 35 minutes at 30°C.

Manning A., Highland H.M., Gasser J., Sim X., Tukiainen T., Fontanillas P., Grarup N., Rivas M.A., Mahajan A., Locke A.E., Cingolani P., Pers T.H., Viñuela A., Brown A.A., Wu Y., Flannick J., Fuchsberger C., Gamazon E.R., Gaulton K.J., Im H.K., Teslovich T.M., Blackwell T.W., Bork-Jensen J., Burtt N.P., Chen Y., Green T., Hartl C., Kang H.M., Kumar A., Ladenvall C., Ma C., Moutsianas L., Pearson R.D., Perry J.R., Rayner N.W., Robertson N.R., Scott L.J., van de Bunt M., Eriksson J.G., Jula A., Koskinen S., Lehtimäki T., Palotie A., Raitakari O.T., Jacobs S.B., Wessel J., Chu A.Y., Scott R.A., Goodarzi M.O., Blancher C., Buck G., Buck D., Chines P.S., Gabriel S., Gjesing A.P., Groves C.J., Hollensted M., Huyghe J.R., Jackson A.U., Jun G., Justesen J.M., Mangino M., Murphy J., Neville M., Onofrio R., Small K.S., Stringham H.M., Trakalo J., Banks E., Carey J., Carneiro M.O., DePristo M., Farjoun Y., Fennell T., Goldstein J.I., Grant G., Hrabé de Angelis M., Maguire J., Neale B.M., Poplin R., Purcell S., Schwarzmayr T., Shakir K., Smith J.D., Strom T.M., Wieland T., Lindstrom J., Brandslund I., Christensen C., Surdulescu G.L., Lakka T.A., Doney A.S., Nilsson P., Wareham N.J., Langenberg C., Varga T.V., Franks P.W., Rolandsson O., Rosengren A.H., Farook V.S., Thameem F., Puppala S., Kumar S., Lehman D.M., Jenkinson C.P., Curran J.E., Hale D.E., Fowler S.P., Arya R., DeFronzo R.A., Abboud H.E., Syvänen A.C., Hicks P.J., Palmer N.D., Ng M.C., Bowden D.W., Freedman B.I., Esko T., Mägi R., Milani L., Mihailov E., Metspalu A., Narisu N., Kinnunen L., Bonnycastle L.L., Swift A., Pasko D., Wood A.R., Fadista J., Pollin T.I., Barzilai N., Atzmon G., Glaser B., Thorand B., Strauch K., Peters A., Roden M., Müller-Nurasyid M., Liang L., Kriebel J., Illig T., Grallert H., Gieger C., Meisinger C., Lannfelt L., Musani S.K., Griswold M., Taylor HA J.r., Wilson G S.r., Correa A., Oksa H., Scott W.R., Afzal U., Tan S.T., Loh M., Chambers J.C., Sehmi J., Kooner J.S., Lehne B., Cho Y.S., Lee J.Y., Han B.G., Käräjämäki A., Qi Q., Qi L., Huang J., Hu F.B., Melander O., Orho-Melander M., Below J.E., Aguilar D., Wong T.Y., Liu J., Khor C.C., Chia K.S., Lim W.Y., Cheng C.Y., Chan E., Tai E.S., Aung T., Linneberg A., Isomaa B., Meitinger T., Tuomi T., Hakaste L., Kravic J., Jørgensen M.E., Lauritzen T., Deloukas P., Stirrups K.E., Owen K.R., Farmer A.J., Frayling T.M., O'Rahilly S.P., Walker M., Levy J.C., Hodgkiss D., Hattersley A.T., Kuulasmaa T., Stančáková A., Barroso I., Bharadwaj D., Chan J., Chandak G.R., Daly M.J., Donnelly P.J., Ebrahim S.B., Elliott P., Fingerlin T., Froguel P., Hu C., Jia W., Ma R.C., McVean G., Park T., Prabhakaran D., Sandhu M., Scott J., Sladek R., Tandon N., Teo Y.Y., Zeggini E., Watanabe R.M., Koistinen H.A., Kesaniemi Y.A., Uusitupa M., Spector T.D., Salomaa V., Rauramaa R., Palmer C.N., Prokopenko I., Morris A.D., Bergman R.N., Collins F.S., Lind L., Ingelsson E., Tuomilehto J., Karpe F., Groop L., Jørgensen T., Hansen T., Pedersen O., Kuusisto J., Abecasis G., Bell G.I., Blangero J., Cox N.J., Duggirala R., Seielstad M., Wilson J.G., Dupuis J., Ripatti S., Hanis C.L., Florez J.C., Mohlke K.L., Meigs J.B., Laakso M., Morris A.P., Boehnke M., Altshuler D., McCarthy M.I., Gloyn A.L, & Lindgren C.M. (2017). A Low-Frequency Inactivating AKT2 Variant Enriched in the Finnish Population Is Associated With Fasting Insulin Levels and Type 2 Diabetes Risk. Diabetes, 66(7), 2019-2032.

+ Open protocol

+ Expand

8

In Vitro Binding and Kinase Assays

Check if the same lab product or an alternative is used in the 5 most similar protocols

GST-CDK4 was purchased from Abcam, Inc (Cambridge, MA), while GST-CDK4/cyclin D1 was from Signal Chem, Inc (Richmond, Canada). For GST protein, E. coli. harboring GST or GST-CDK4 were grown overnight in room temperature. The recombinant proteins were induced with 1 mM IPTG for 4 hours. GST was purified by glutathione column and diluted to 1 ng/ul with 1X kinase assay buffer provided by the manufacturer (Cell Signaling, Inc, Danvers, MA). This was followed by combining 25 μl GST or GST-CDK4 with 1 ng substrate (GST-Rb) with or without 25 ng of cyclin D1, or HisTAG-MCM8, or HisTAG- ΔMCM8^del261-290 or 2 ng of HisTAG-MCM8CBM in 1xkinase reaction buffer. The solutions were incubated at 37°C for 60 minutes. The reactions were terminated by adding 25 μl of 2N NaOH stop solution to each reaction well. The kinase activities were quantified using the kit and protocols of ADP-Glo™ Kinase Assay from Promega, Inc, Madison, WI.
For in vitro MCM8/cyclin D1/CDK4 binding assay, the following condition was used: 25 ng of each of HisTAG-MCM8, GST-cyclinD1 and GST-CDK4 produced from E.coli. were incubated with 150 mM NaCl, 25 mM MOPS, pH 7.2, 12.5 mM MgCl₂, 12.5 mM β-glycerol phosphate at 37°C for 2 h. The products were then resolved in 6% non-denaturing polyacrylamide gel electrophoresis.

He D.M., Ren B.G., Liu S., Tang L.Z., Cieply K., Tseng G., Yu Y.P, & Luo J.H. (2017). Oncogenic Activity of Amplified Miniature Chromosome Maintenance 8 in Human Malignancies. Oncogene, 36(25), 3629-3639.

+ Open protocol

+ Expand

9

Kinase-mediated Phosphorylation of Bid

Check if the same lab product or an alternative is used in the 5 most similar protocols

Kinase reactions were performed in 100 μl of kinase assay buffer (Cell Signaling Technology) using 5 μg activated recombinant JNKs or 500 μl of lysate from ALDH1L1-transfected cells, and 2 μg purified human recombinant Bid. Reaction was initiated by 1 mM ATP. Phosphorylation of Bid at T59 was evaluated by immunoblot assays with a specific antibody after SDS-PAGE. For the LC/MS-MS analysis of phosphorylated Bid peptides, after the kinase reaction with pure JNKs reaction mixture has been subjected to SDS-PAGE. In the case of kinase reaction with cell lysates, Bid was pulled down on Ni-beads and eluted with 250 mM imidazole prior to SDS-PAGE.

Prakasam A., Ghose S., Oleinik N.V., Bethard J.R., Peterson Y.K., Krupenko N.I, & Krupenko S.A. (2014). JNK1/2 regulate Bid by direct phosphorylation at Thr59 in response to ALDH1L1. Cell Death & Disease, 5(7), e1358-.

+ Open protocol

+ Expand

10

Kinase Assay for JAK2-Mediated GSK3β Phosphorylation

Cited 1 time

Check if the same lab product or an alternative is used in the 5 most similar protocols

The cytosolic and nuclear fractions of cells were prepared as previously described (Wu et al. 2008 (link); Gong et al. 2014 (link)). Immunoprecipitation and western blot analyses were performed using standard protocols as previously described (Wu et al. 2008 (link)). The in vitro kinase assay to assess the phosphorylation of GSK3β by JAK2 was performed as previously described (Wu et al. 2008 (link)). To validate JAK2 as a kinase phosphorylating GSK3β at Y216, recombinant human active JAK2 (Abcam, ab42621) and human GSK3β (Abcam, ab63193) were used in the in vitro kinase assay. 30 ng of GSK3β was incubated with 50 μl kinase assay buffer (Cell Signaling) in the presence or absence of 30 ng active JAK2 and 2 mM ATP at 37 °C for 30 min. The reactive mixture was subjected to western analyses with antibodies recognizing p-Tyr216-GSK3β, GSK3β, and JAK2. For western, GAPDH or β-actin was used as the internal standard of total target proteins, and phosphorylated proteins were normalized to total proteins, respectively. Immunoreactive bands from duplicates or triplicates were quantified by ImageJ, and the mean intensity from first band was set to 1.

Shi W., Xu C., Gong Y., Wang J., Ren Q., Yan Z., Mei L., Tang C., Ji X., Hu X., Qv M., Hussain M., Zeng L.H, & Wu X. (2021). RhoA/Rock activation represents a new mechanism for inactivating Wnt/β-catenin signaling in the aging-associated bone loss. Cell Regeneration, 10, 8.

+ Open protocol

+ Expand

Kinase assay buffer

Lab products found in correlation

11 protocols using kinase assay buffer

Characterizing IKK2 Complex Activation

RASSF9 Phosphorylation by TAK1 Kinase

In Vitro Kinase Assay for IKKβ

Kinase activity of Akt with USP14

In Vitro Binding and Kinase Assays

IKK Complex Kinase Assay Protocol

AKT2 Variant Kinase Assay

In Vitro Binding and Kinase Assays

Kinase-mediated Phosphorylation of Bid

Kinase Assay for JAK2-Mediated GSK3β Phosphorylation

About PubCompare

Ready to get started?