Clone 9e10, by Merck Group - Product details

1

Quantifying Anti-CD19 scFv Binding

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2x10⁵ K562/CD19 cells were incubated for 1 h at 4°C with the indicated concentration of His-tagged anti-CD19 scFvs for binding assays and with a mixture of 10 nM myc-tagged FMC63 scFv and the indicated concentration of His-tagged human scFvs for competition binding assays, respectively. Following two washes, cells were stained with an anti-His mAb (Qiagen, #35370) for binding assays or an anti-myc mAb (EMD Millipore, clone 9E10) for competition binding assays. Mean fluorescence intensity was measured using a Guava flow cytometer (EMD Millipore).

Sommermeyer D., Hill T., Shamah S.M., Salter A.I., Chen Y., Mohler K.M, & Riddell S.R. (2017). Fully human CD19-specific chimeric antigen receptors for T-cell therapy. Leukemia, 31(10), 2191-2199.

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2

Quantifying Anti-CD19 scFv Binding

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2x10⁵ K562/CD19 cells were incubated for 1 h at 4°C with the indicated concentration of His-tagged anti-CD19 scFvs for binding assays and with a mixture of 10 nM myc-tagged FMC63 scFv and the indicated concentration of His-tagged human scFvs for competition binding assays, respectively. Following two washes, cells were stained with an anti-His mAb (Qiagen, #35370) for binding assays or an anti-myc mAb (EMD Millipore, clone 9E10) for competition binding assays. Mean fluorescence intensity was measured using a Guava flow cytometer (EMD Millipore).

Sommermeyer D., Hill T., Shamah S.M., Salter A.I., Chen Y., Mohler K.M, & Riddell S.R. (2017). Fully human CD19-specific chimeric antigen receptors for T-cell therapy. Leukemia, 31(10), 2191-2199.

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3

Co-immunoprecipitation of Protein Complexes

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For co-immunoprecipitation (co-IP), cell lysates were prepared by adding lysis buffer (150 mM NaCl, 1% IGEPAL® CA-630, 50 mM Tris·Cl; pH 8.0) supplemented with a protease inhibitor cocktail (Roche, Basel, Switzerland). The lysate was immunoprecipitated using 2–3 μg of antibody (specificity indicated in the figures), mouse immunoglobulin G (IgG; Sigma-Aldrich, St. Louis, MO, USA), and incubated with 50 μL of Protein G-Sepharose (GE Healthcare, Chicago, IL, USA). The immunoprecipitates were washed three times in 1 mL of ice-cold lysis buffer, followed by additional wash an additional time with 1 mL of 50 mM Tris·Cl (pH 8.0). The precipitated proteins were separated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (8%–12%). For western blot analysis, the blots were incubated using the antibody indicated in the figures. All co-IPs and western blot analyses were performed more than twice to confirm that the data were reproducible. The following antibodies were used in the co-IPs and western blot analyses: monoclonal anti-FLAG antibody (1:2000, Clone M2; Sigma-Aldrich), monoclonal anti-HA antibody (1:2000, Clone HA-7; Sigma-Aldrich), and monoclonal anti-Myc antibody (1:2000, Clone 9E10; Sigma-Aldrich).

Yoo K.S., Lee K., Oh J.Y., Lee H., Park H., Park Y.S, & Kim H.K. (2019). Postsynaptic density protein 95 (PSD-95) is transported by KIF5 to dendritic regions. Molecular Brain, 12, 97.

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4

Protein Extraction and Western Blotting

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Equivalent optical densities (1 OD 600 nm) of cells were lysed in cold TCA buffer (10 mM Tris⋅HCl pH 8.0, 10% trichloroacetic acid, 25 mM NH₄OAc, 1 mM Na₂ ethylenediaminetetraacetic acid) and incubated on ice. Following centrifugation to clarify lysates, pellets were resuspended in resuspension solution (0.1 M Tris⋅HCl pH 11.0, 3% sodium dodecyl sulfate [SDS]) and boiled at 95 °C for 5 min. Clarified samples were centrifuged and the supernatants were transferred to new tubes containing 4× sample buffer (250 mM Tris⋅HCl pH 6.8, 8% SDS, 40% glycerol, 20% β-mercaptoethanol) and boiled at 95 °C for an additional 5 min. Western blotting was performed using antibodies against Hog1 (sc-165978, Santa Cruz Biotechnology), phosphorylated Hog1 (anti-phospho-p38 MAPK, 9211L; Cell Signaling Technology), GFP (clone JL8,63268, Clontech), V5 (R96025, Invitrogen), MYC (clone 9E10, M5546; Sigma), PSTAIRE (P7962, Sigma), and G6PDH (A9521, Sigma).

Flynn M.J, & Benanti J.A. (2022). Cip1 tunes cell cycle arrest duration upon calcineurin activation. Proceedings of the National Academy of Sciences of the United States of America, 119(23), e2202469119.

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5

Immunoblotting Antibody Validation

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Antibodies to MYH1 (1:3000; Noq7 and M8421; Sigma-Aldrich), NURR1 (1:1000; Abcam, ab41917), MEF2 (1:1000; Santa Cruz Biotechnology, sc-313), GFP (1:1000; Life Technology, A11122), Flag (1:1000; clone M2; Sigma-Aldrich), MYC (1:3000; clone 9E10; Sigma-Aldrich), and GAPDH (1:8000; Millipore, MAB374) and goat anti-mouse and goat-anti rabbit HRP-conjugated secondary antibodies (1:3000; Bio-Rad) were used for the described experiments.

Amoasii L., Holland W., Sanchez-Ortiz E., Baskin K.K., Pearson M., Burgess S.C., Nelson B.R., Bassel-Duby R, & Olson E.N. (2016). A MED13-dependent skeletal muscle gene program controls systemic glucose homeostasis and hepatic metabolism. Genes & Development, 30(4), 434-446.

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6

Detecting Ubiquitinated c-Myc Protein

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Human HCT116 14-3-3σ^−/−, MDA-MB-231, HCT116 p53^−/− and H1299 cells carrying retroviral a tet-On Flag-14-3-3σ system were used for the assay. 10 mM MG132 (Sigma-Aldrich (St Louis, MO, USA), Catalog # M7449) was added to the culture media for 8 hours for blocking proteasome-mediated protein degradation^{57 ,58} to facilitate the accumulation of ubiquitinated proteins. NP-40-containing lysis buffer was used for cell lysis. 5 mM N-Ethylmaleimide (Sigma-Aldrich, Catalog # E3876) was added to NP40-containing lysis buffer to inhibit deubiquitinases and prevent deubiquitination. The ubiquitinated c-Myc protein was immunoprecipitated with monoclonal anti-c-Myc antibody (Sigma-Aldrich, 9E10 clone, Catalog # M4439) and subsequently immunoblotted with monoclonal mouse anti-ubiquitin antibody (Life Technologies, USA, Catalog #13-1600).

Phan L., Chou P.C., Velazquez-Torres G., Samudio I., Parreno K., Huang Y., Tseng C., Vu T., Gully C., Su C.H., Wang E., Chen J., Choi H.H., Fuentes-Mattei E., Shin J.H., Shiang C., Grabiner B., Blonska M., Skerl S., Shao Y., Cody D., Delacerda J., Kingsley C., Webb D., Carlock C., Zhou Z., Hsieh Y.C., Lee J., Elliott A., Ramirez M., Bankson J., Hazle J., Wang Y., Li L., Weng S., Rizk N., Wen Y.Y., Lin X., Wang H., Wang H., Zhang A., Xia X., Wu Y., Habra M., Yang W., Pusztai L., Yeung S.C, & Lee M.H. (2015). The cell cycle regulator 14-3-3σ opposes and reverses cancer metabolic reprogramming. Nature communications, 6, 7530.

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7

Detecting Ubiquitinated c-Myc Protein

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Human HCT116 14-3-3σ^−/−, MDA-MB-231, HCT116 p53^−/− and H1299 cells carrying retroviral a tet-On Flag-14-3-3σ system were used for the assay. 10 mM MG132 (Sigma-Aldrich (St Louis, MO, USA), Catalog # M7449) was added to the culture media for 8 hours for blocking proteasome-mediated protein degradation^{57 ,58} to facilitate the accumulation of ubiquitinated proteins. NP-40-containing lysis buffer was used for cell lysis. 5 mM N-Ethylmaleimide (Sigma-Aldrich, Catalog # E3876) was added to NP40-containing lysis buffer to inhibit deubiquitinases and prevent deubiquitination. The ubiquitinated c-Myc protein was immunoprecipitated with monoclonal anti-c-Myc antibody (Sigma-Aldrich, 9E10 clone, Catalog # M4439) and subsequently immunoblotted with monoclonal mouse anti-ubiquitin antibody (Life Technologies, USA, Catalog #13-1600).

Phan L., Chou P.C., Velazquez-Torres G., Samudio I., Parreno K., Huang Y., Tseng C., Vu T., Gully C., Su C.H., Wang E., Chen J., Choi H.H., Fuentes-Mattei E., Shin J.H., Shiang C., Grabiner B., Blonska M., Skerl S., Shao Y., Cody D., Delacerda J., Kingsley C., Webb D., Carlock C., Zhou Z., Hsieh Y.C., Lee J., Elliott A., Ramirez M., Bankson J., Hazle J., Wang Y., Li L., Weng S., Rizk N., Wen Y.Y., Lin X., Wang H., Wang H., Zhang A., Xia X., Wu Y., Habra M., Yang W., Pusztai L., Yeung S.C, & Lee M.H. (2015). The cell cycle regulator 14-3-3σ opposes and reverses cancer metabolic reprogramming. Nature communications, 6, 7530.

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Clone 9e10

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7 protocols using clone 9e10

Quantifying Anti-CD19 scFv Binding

Quantifying Anti-CD19 scFv Binding

Co-immunoprecipitation of Protein Complexes

Protein Extraction and Western Blotting

Immunoblotting Antibody Validation

Detecting Ubiquitinated c-Myc Protein

Detecting Ubiquitinated c-Myc Protein

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