Ab138911

Manufactured by Abcam

Ab138911 is a lab equipment product offered by Abcam. It serves as a core component in experimental procedures. The description of this product remains concise and factual, without interpretation or extrapolation on its intended use.

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Lab products found in correlation

Ab138910, by Abcam (10 mentions) Ab92570, by Abcam (6 mentions) Anti ha agarose antibody, by Merck Group (2 mentions) Ab55695, by Abcam (2 mentions) Kinase reaction and alkylation protocol, by Abcam (1 mentions) Anti p44 42 erk antibody, by Cell Signaling Technology (1 mentions) Anti myc sc 40, by Santa Cruz Biotechnology (1 mentions) Anti phospho yap s127 4911s, by Cell Signaling Technology (1 mentions) Anti flag m2 f3165 5mg, by Merck Group (1 mentions) Anti yap sc 101199, by Santa Cruz Biotechnology (1 mentions) P nitrobenzyl mesylate, by Abcam (1 mentions) Anti α tubulin t6199, by Merck Group (1 mentions) Nitrocellulose membrane, by GE Healthcare (1 mentions) Atp γ s, by Abcam (1 mentions)

10 protocols using ab138911

CK2α Kinase Assay of G6PD Phosphorylation

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Kinase reactions were performed as described previously [11 (link)]. Purified CK2α protein (10 ng) was incubated with purified G6PD protein (200 ng) in 25 μL kinase buffer (50 mM Tris-HCl, pH 7.5, 100 mM KCl, 5 mM MgCl₂, 1 mM Na₃VO₄, 50 μM DTT, 5% glycerol, 100 μM ATP) at 30 °C for 30 min. For in vitro kinase assay using [γ-³²P]-ATP, 10 μCi of the ³²P-labeled ATP was added in the reaction system. G6PD protein was precipitated and analyzed by SDS-PAGE, followed by immunoblot with indicated phosphorylation site-specific antibodies or autoradiography.
For in vitro kinase assay using ATPγS, the experiment was performed following the Kinase Reaction and Alkylation Protocol (Abcam) according to previous report [12 ]. Briefly, purified CK2α protein and G6PD protein were incubated with the reaction buffer (20 mM Hepes, pH 7.5, 100 mM NaCl, 1 mM ATPγS (ab138911, Abcam), 10 mM MgCl₂) for 30 min at 30 °C, followed by addition of 2.5 mM p-nitrobenzyl mesylate (ab138910, Abcam) to the reaction system and incubation at room temperature for another 1 h G6PD protein was then precipitated and analyzed by SDS-PAGE, followed by immunoblot with the thiophosphate ester specific antibody (ab92570, Abcam).

Hao Y., Ren T., Huang X., Li M., Lee J.H., Chen Q., Liu R, & Tang Q. (2023). Rapid phosphorylation of glucose-6-phosphate dehydrogenase by casein kinase 2 sustains redox homeostasis under ionizing radiation. Redox Biology, 65, 102810.

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In vitro CDH1 Kinase Assay

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In vitro kinase assay was performed as previously described^{84 (link)}. Briefly, HA-tagged CDH1 WT and mutants were transfected into HEK293T cells, followed by being immunoprecipitated with monoclonal Anti-HA-Agarose antibody (Sigma, A2095). The purified HA-CDH1 proteins were then incubated with 500 uM of ATPγS (Abcam, ab138911) and 0.5 ug of recombinant human cyclin D1+CDK4 proteins (Abcam, ab55695) in the kinase reaction buffer (50mM Tris-HCl, 10mM MgCl₂, 0.1mM EDTA, 2mM DTT, 0.01% Brij 35, pH 7.5) for 30 min at room temperature. Then adding 2 mM of PNBM (Abcam, ab138910) and allowing the alkylating reaction proceed for additional 2h at room temperature. The reaction was then terminated by adding 5x SDS loading buffer and boiled for 10 min. Samples were then subjected to IB using anti-Thiophosphate ester antibody (Abcam, ab92570).

Ke S., Dang F., Wang L., Chen J.Y., Naik M.T., Thavamani A., Liu Y., Li W., Kim N., Naik N.M., Sui H., Tang W., Qiu C., Koikawa K., Batalini F., Wang X., Clohessy J.G., Heng Y.J., Lahav G., Gray N.S., Zho X.Z., Wei W., Wulf G.M, & Lu K.P. (2023). Reciprocal inhibition of PIN1 and APC/CCDH1 controls timely G1/S transition and creates therapeutic vulnerability. Research Square.

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CDH1 Phosphorylation Assay

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HA-tagged CDH1 WT and mutants were transfected into HEK293T cells, followed by being immunoprecipitated with monoclonal Anti-HA-Agarose antibody (Sigma, A2095). The purified HA-CDH1 proteins were then incubated with 500 uM of ATPγS (Abcam, ab138911) and 0.5 ug of recombinant human cyclin D1 + CDK4 proteins (Abcam, ab55695) in the kinase reaction buffer (50 mM Tris-HCl, 10 mM MgCl₂, 0.1 mM EDTA, 2 mM DTT, 0.01% Brij 35, pH 7.5) for 30 min at room temperature. Then adding 2 mM of PNBM (Abcam, ab138910) and allowing the alkylating reaction proceed for additional 2 h at room temperature. The reaction was then terminated by adding 5x SDS loading buffer and boiled for 10 min. Samples were then subjected to IB using anti-Thiophosphate ester antibody (Abcam, ab92570).

Ke S., Dang F., Wang L., Chen J.Y., Naik M.T., Li W., Thavamani A., Kim N., Naik N.M., Sui H., Tang W., Qiu C., Koikawa K., Batalini F., Stern Gatof E., Isaza D.A., Patel J.M., Wang X., Clohessy J.G., Heng Y.J., Lahav G., Liu Y., Gray N.S., Zhou X.Z., Wei W., Wulf G.M, & Lu K.P. (2024). Reciprocal antagonism of PIN1-APC/CCDH1 governs mitotic protein stability and cell cycle entry. Nature Communications, 15, 3220.

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In Vitro Phosphorylation Assay for MAPK Signaling

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In vitro phosphorylation assays were performed as previously described [57 (link)]. Recombinant glutathione S-transferase (GST)-OsMKK6 or GST-OsMKK6^DD (500 ng) was incubated with the substrate proteins maltose binding protein (MBP)-OsMPK4 or MBP-OsMPK4m (1 μg) in kinase reaction buffer (30 mM Tris-HCl pH 7.5, 1 mM EGTA, 10 mM MgCl₂, 1 mM DTT, and 3 μL 10 mM ATPγS [Abcam, ab138911]) at 30 °C for 30 min. After adding 1.5 μL 50 mM p-Nitrobenzyl mesylate (PNBM, Abcam, Cambridge, UK; ab138910) to the reactions, the samples were incubated at 30 °C for 1.5 h. Recombinant proteins were separated by 10% (w/v) SDS-PAGE, and phosphorylated recombinant MKK6, MKK6^DD, MPK4, and MPK4m were detected by immunoblotting with anti-thiophosphate ester rabbit monoclonal antibodies (Abcam, Cambridge, UK; ab92570, 1:5000).
To detect MAPK activation, WT and rsr25 plants were inoculated with Xoo, and total proteins were extracted from the plants at 0 h, 48 h, 96 h, and 120 h after inoculation. MPK activation was detected by immunoblotting with an anti-p44/42 ERK antibody (Cell Signaling, Danvers, MA, USA; 4370S).

Jiang R., Zhou S., Da X., Yan P., Wang K., Xu J, & Mo X. (2023). OsMKK6 Regulates Disease Resistance in Rice. International Journal of Molecular Sciences, 24(16), 12678.

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Western Blot Antibody Characterization

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For Western blotting, anti-α-tubulin (T6199, 1:5000 dilution) and anti-Flag (M2) (F3165-5MG, 1:5000 dilution) antibodies were obtained from Sigma-Aldrich. Anti-Myc (sc-40, 1:500 dilution) and anti-GST antibodies (sc-138, 1:1000 dilution) were purchased from Santa Cruz Biotechnology. Anti-phospho-YAP (S127) (4911S, 1:1000 dilution) and anti-phospho-LATS1 (Thr1079) (8654S, 1:1000 dilution) antibodies were purchased from Cell Signaling Technology. Anti-hemagglutinin (HA) monoclonal antibody (901514, 1:2000 dilution) was obtained from BioLegend. Anti-Thiophosphate ester antibody (ab92670, 1:1000 dilution) was purchased from Abcam. The YAP and MBP polyclonal antibodies were generated as previously described^{57 (link),61 (link)}. ATP-γ-S kinase substrate (ab138911) and p-Nitrobenzyl mesylate (ab138910) were obtained from Abcam. For immunostaining, an anti-YAP (sc-101199, 1:200 dilution) monoclonal antibody was purchased from Santa Cruz Biotechnology. Anti-Flag polyclonal antibody (F7425, 1:5000 dilution) was obtained from Sigma-Aldrich.

Chen Y., Han H., Seo G., Vargas R.E., Yang B., Chuc K., Zhao H, & Wang W. (2020). Systematic analysis of the Hippo pathway organization and oncogenic alteration in evolution. Scientific Reports, 10, 3173.

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TTBK2 Kinase Assay with CEP83

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Various FH-TTBK2 proteins were transiently expressed in 293T cells for 48 h and immunoprecipitated by M2 beads. Beads were washed three times with TBS buffer (50 mM Tris-HCl and 150 mM NaCl, pH 7.4) followed by TTBK2 kinase reaction buffer (50 mM Tris, pH 7.4, and 10 mM MgCl₂) before performing the TTBK2 kinase assay. The TTBK2 kinase assay was performed by incubating TTBK2 protein on the beads with 5 µg recombinant His-CEP83^WT or His-CEP83^4A for 30 min at RT in 30 µl kinase reaction buffer that contained 100 ng ATP-gamma-S (ab138911; Abcam). After incubation, 1 µl of 50 mM p-nitrobenzyl mesylate (ab138910; Abcam) was added to the kinase reaction for 90 min at RT to stop the kinase reaction (Allen et al., 2007 (link)). Proteins were separated by SDS-PAGE and then transferred to the nitrocellulose membranes (GE Healthcare) for Western blot. The immunoreactive bands were detected by Odyssey Image Systems (LI-COR). The gel was also stained with Coomassie brilliant blue (CBB) to ascertain migration of the proteins. For the TTBK2 in vitro kinase assay that used GST-TTBK2 (1–622)^WT and GST-TTBK2 (aa 1–622)^KD purified from bacteria, 0.5 µg GST-TTBK2 (aa 1–622)^WT or GST-TTBK2(aa 1–622)^KD and 5 µg CEP83 were added in the 30 µl of kinase reaction buffer that contained 100 ng ATP-gamma-S for 30 min at room temperature.

Lo C.H., Lin I.H., Yang T.T., Huang Y.C., Tanos B.E., Chou P.C., Chang C.W., Tsay Y.G., Liao J.C, & Wang W.J. (2019). Phosphorylation of CEP83 by TTBK2 is necessary for cilia initiation. The Journal of Cell Biology, 218(10), 3489-3505.

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PD-1 kinase assay with ERK1

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Recombinant human GST-PD-1 (23-170), PD-1 (192-288) WT, and T234A mutant proteins were purified from E. coli BL21 cells. 0.1 µg human active ERK1 (E7407, Sigma) was incubated with 1 μg recombinant GST or GST-PD-1 truncates in the kinase assay buffer (25 mM Tris-HCl at pH 7.5, 25 mM KCl, 5 mM MgCl₂) with 500 μM ATP-γ-S (ab138911, Abcam) and 1 mM DTT for 1 h at 30 °C. The reaction was stopped by adding 2 μl EDTA (0.5 M) and were further incubated with 0.5 mM p-Nitrobenzyl mesylate (PNBM) (ab138910, Abcam) for 1 h at 30 °C. Subsequently, the signal of phosphorylation was detected by immunoblotting with anti-thiophosphate ester antibody (ab92570, Abcam).

Xiao X., Shi J., He C., Bu X., Sun Y., Gao M., Xiang B., Xiong W., Dai P., Mao Q., Xing X., Yao Y., Yu H., Xu G., Li S., Ren Y., Chen B., Jiang C., Meng G., Lee Y.R., Wei W., Freeman G.J., Xie C, & Zhang J. (2023). ERK and USP5 govern PD-1 homeostasis via deubiquitination to modulate tumor immunotherapy. Nature Communications, 14, 2859.

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In Vitro Kinase Assay with Thiophosphorylation

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The immunoprecipitated lysates were washed with PLB buffer three times and kinase buffer (20 mM Hepes pH 7.4, 50 mM KoAC, 1 mM MnCL₂, 1 mM MgCl₂, 1 mM Na₂Mao₄, and 2 mM NaF) once, followed by incubation in kinase reaction mix (kinase buffer with ATPγS- Kinase substrate, ab138911; Abcam) for 30 min at 30°C. EDTA (0.5 M, pH 8.0) (R1021; Thermo Fisher Scientific) was used to stop reaction. P-nitrobenzyl mesylate (PNBM , ab138910; Abcam) was added and incubated for 45 min at 25°C to generate thiophosphate esters on the thiophosphorylated substrates in the in vitro kinase assay, as previously shown (33 ). Next day, beads were washed with PLB and boiled after adding 5× SDS loading buffer for 5 min at 95°C. Proteins were then separated by electrophoresis in SDS-PAGE gels for analysis.

Yildirim A.D., Citir M., Dogan A.E., Veli Z., Yildirim Z., Tufanli O., Traynor-Kaplan A., Schultz C, & Erbay E. (2022). ER Stress-Induced Sphingosine-1-Phosphate Lyase Phosphorylation Potentiates the Mitochondrial Unfolded Protein Response. Journal of Lipid Research, 63(10), 100279.

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Kinase Activity Assay with ATP-γ-S Labeling

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Kinase assays were performed as previously described (50 (link)). Briefly, SFB-tagged proteins were expressed in HEK293T cells for 48 hours. Cells were lysed in NETN buffer with protease inhibitors, 10 mM MgCl₂, and Turbonuclease for 1 hour at 4°C. Samples were centrifuged at 14,000×g, 4°C for 20 minutes. Supernatants were incubated with S-protein beads for 1 hour at 4°C. Beads were washed for three times with NETN, twice with kinase wash buffer (40 mM HEPES pH 7.5, 250 mM NaCl), and once with kinase reaction buffer (30 mM HEPES pH 7.5, 50 mM potassium acetate, 5 mM MgCl₂). Equal volumes of SFB-tagged proteins in kinase reaction buffer and 500 μM ATP-γ-S (Abcam, ab138911) were mixed and incubated at 30°C for 30 minutes. ATP- γ-S was alkylated by p-Nitrobenzyl mesylate (PNBM, Abcam, ab138910) to a final concentration of 2.5 mM and incubated at room temperature for 1 hour. Alkylation was terminated using 2x Laemmli buffer. Phosphorylation was detected after Western blotting by an anti-thiophosphate ester antibody.

West K.L., Kreiling N., Raney K.D., Ghosal G, & Leung J.W. (2024). Autophosphorylation of the Tousled-like kinases TLK1 and TLK2 regulates recruitment to damaged chromatin via PCNA interaction. bioRxiv.

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Kinase Reaction Assay for AMPK

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In [γS] ATP-containing kinase reaction, 1 μg of GST-GD-NT purified from HEK293 cells transfected with the indicated constructs, 300 ng of AMPK complex prepared from the HEK293 cells transfected with the indicated constructs, 1 mM [γS] ATP (ab138911, Abcam) were added in kinase reaction buffer (50 mM Tris (pH 7.5), 1 μM MnCl₂, 2 mM dithiothreitol (DTT)). After 30 min of incubation at 30 °C, the reaction was subsequently stopped by adding in 0.1 mM EDTA, and further reacted for another 1 h by adding pNitrobenzyl mesylate (ab138910, Abcam) to alkylate the thiophosphorylation site on the substrates. The reaction was stopped by adding SDS loading buffer and resolved by SDS–PAGE. Phosphorylation of GD-NT was detected using anti-Thiophosphate ester (ab92570, Abcam).
In regular ATP-containing kinase reaction, 1 μg of GST-GD-NT purified from HEK293 cells transfected with the indicated constructs, 300 ng of AMPK complex prepared from the HEK293 cells transfected with the indicated constructs or commercially obtained from Sigma (14-840), 200 μM regular ATP were added in kinase reaction buffer (50 mM Tris (pH 7.5), 1 μM MnCl₂, 2 mM dithiothreitol (DTT)). After 30 minutes of incubation at 30 °C, the reaction was stopped by adding SDS loading buffer and resolved by SDS–PAGE. GD-NT phosphorylation was detected using the antibody pS46-GD.

Chu X., Xiao X., Wang G., Uosef A., Lou X., Arnold P., Wang Y., Kong G., Wen M., Minze L.J, & Li X.C. (2023). Gasdermin D-mediated pyroptosis is regulated by AMPK-mediated phosphorylation in tumor cells. Cell Death & Disease, 14(7), 469.

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