Total her2

Manufactured by Cell Signaling Technology

Sourced in United States

The Total HER2 product is a laboratory tool used to quantify the total amount of HER2 protein in a sample. It provides a measurement of the overall HER2 expression levels, without interpreting the intended use or clinical implications.

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Lab products found in correlation

6 protocols using total her2

Quantifying EGFR and HER2 Activation

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Protein was harvested from the parental Ba/F3 cell line and each of the Ba/F3 exon 20 mutants found to be activating mutations as described above. ELISA was performed following the manufacturer’s instructions for total EGFR (Cell signaling, no. 7250) and total HER2 (Cell Signaling, no. 7310). Relative expression determined through ELISA was plotted against IC₅₀ values calculated as described above. Pearson correlations and P values were determined using GraphPad Prism.

Robichaux J.P., Elamin Y.Y., Tan Z., Carter B.W., Zhang S., Liu S., Li S., Chen T., Poteete A., Estrada-Bernal A., Le A.T., Truini A., Nilsson M.B., Sun H., Roarty E., Goldberg S.B., Brahmer J.R., Altan M., Lu C., Papadimitrakopoulou V., Politi6 K., Doebele R.C., Wong K.K, & Heymach J.V. (2018). Mechanisms and Clinical Activity of an EGFR and HER2 Exon 20-selective Kinase Inhibitor in Non-small Cell Lung Cancer. Nature medicine, 24(5), 638-646.

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Signaling Pathways in Cancer Cell Lines

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NRG1 was purchased from PeproTech. Lapatinib was a kind gift from Professor György Kéri (Vichem Chemie Research Ltd Hungary). Bosutinib was purchased from LC Labs. Total HER2, HER3, PKB, HER2 pY877, HER3 pY1289, PKB pS473 and ERK1/2 pT202/pY204 antibodies were purchased from Cell Signaling Technology, anti-α-tubulin from Sigma, total ERK1/2 from Merck, and Alexa Fluor-488 conjugated anti-HER3 antibody from R&D systems.

Claus J., Patel G., Autore F., Colomba A., Weitsman G., Soliman T.N., Roberts S., Zanetti-Domingues L.C., Hirsch M., Collu F., George R., Ortiz-Zapater E., Barber P.R., Vojnovic B., Yarden Y., Martin-Fernandez M.L., Cameron A., Fraternali F., Ng T, & Parker P.J. (2018). Inhibitor-induced HER2-HER3 heterodimerisation promotes proliferation through a novel dimer interface. eLife, 7, e32271.

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Molecular Profiling of Receptor Tyrosine Kinases

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Entrectinib, crizotinib, capmatinib, and afatinib were purchased from Selleck Chemicals. Antibodies to total AKT, AKT pS473, total ERK, ERK pT202/Y204, total ROS1, ROS1 pY2274, total MET, MET pY1234/1235, total AXL, total EGFR, EGFR pY1068, EGFR pY1086, EGFR pY1173, total HER2, HER2 pY1196, HER2 pY1221/1222, HER3 pY1289 were purchased from Cell Signaling Technologies. Antibody to GAPDH was purchased from Santa Cruz Biotechnologies Inc. MET and CEP7 interphase FISH probes were purchased from Abbott Molecular Inc. MET, CEP7, MYC, and CEP8 metaphase FISH probes were purchased from Empire Genomics.

Tyler L.C., Le A.T., Chen N., Nijmeh H., Bao L., Wilson T.R., Chen D., Simmons B., Turner K.M., Perusse D., Kasibhatla S., Christiansen J., Dudek A.Z, & Doebele R.C. (2022). MET gene amplification is a mechanism of resistance to entrectinib in ROS1+ NSCLC. Thoracic Cancer, 13(21), 3032-3041.

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Western Blot Analysis of Signaling Proteins

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Cells grown under the previously specified conditions were lysed in a Lysis Buffer (Cell Signaling Technology). Western blot analyses were conducted after separation by SDS-PAGE electrophoresis and transfer to polyvinylidene difluoride-P immobilon membranes (Millipore). Immunoblotting was conducted according to the antibody manufacturer’s recommendations. Anti-phospho-Akt (Ser473), total AKT, phospho EGFR (Tyr1068), total EGFR, phospho Erk (Thr202/Tyr204), total Erk, phospho HER2 (Tyr1221/1222), total HER2, and PARP antibodies were obtained from Cell Signaling Technology. Anti-α-Tubulin antibody was purchased from Sigma-Aldrich.

Kosaka T., Tanizaki J., Paranal R.M., Endoh H., Lydon C., Capelletti M., Repellin C.E., Choi J., Ogino A., Calles A., Ercan D., Redig A.J., Bahcall M., Oxnard G.R., Eck M.J, & Jänne P.A. (2017). Response heterogeneity of EGFR and HER2 exon 20 insertions to covalent EGFR and HER2 inhibitors. Cancer research, 77(10), 2712-2721.

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Western Blot Analysis of Signaling Proteins

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Isolation of cell extracts and western blot analysis were described previously [23 (link)]. The protein concentrations were measured by a BCA protein assay kit (Beyotime Biotechnology, Shanghai, China). Immuno-detection was carried out using ER-α36 (Cell Applications, San Diego, CA, USA), p-ERK1/2 (Cell Signaling Technology, Boston, MA, USA) and total-ERK1/2 (Cell Signaling Technology, Boston, MA, USA), p-EGFR (Cell Signaling Technology, Boston, MA, USA) and total-EGFR (Cell Signaling Technology, Boston, MA, USA), p-HER-2 (Cell Signaling Technology, Boston, MA, USA) and total-HER-2 (Cell Signaling Technology, Boston, MA, USA) antibodies or Tubulin antibody. Tubulin (Beyotime, Shanghai, China) was used as a control for equal loading and transfer.

Zhu L., Zou J., Zhao Y., Jiang X., Wang Y., Wang X, & Chen B. (2018). ER-α36 mediates cisplatin resistance in breast cancer cells through EGFR/HER-2/ERK signaling pathway. Journal of Experimental & Clinical Cancer Research : CR, 37, 123.

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Quantitative Protein Detection Assay

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Cell extracts were prepared by detergent lysis RIPA (Sigma) containing 10-mmol/L iodoacetamide (G-Biosciences), protease inhibitor (Sigma), and phosphatase inhibitors (Sigma) cocktails. The soluble protein concentration was determined by micro-BCA assay (Pierce Biotechnology). Protein immunodetection was performed by electrophoretic transfer of SDS-PAGE separated proteins to nitrocellulose, incubation with antibody, and chemiluminescent second step detection. Nitrocellulose membranes were blocked with 5% nonfat dry milk in TBS and incubated overnight with primary antibody in 5% BSA. The following primary antibody from Cell Signaling Technology were used at 1:1,000 dilution: phospho-HER2 and total HER2. β-Actin antibody, used as a control for protein loading, was purchased from Sigma. Horseradish peroxidase–conjugated secondary antibodies (Cell Signaling Technology) were incubated in nonfat dry milk at 1:5,000 dilution for 1 hour. SuperSignal chemiluminescent reagent (Pierce Biotechnology) was used according to the manufacturer's directions, and blots were imaged using the Alpha Innotech image analyzer and AlphaEaseFC software (Alpha Innotech).

Ishiyama N., O'Connor M., Salomatov A., Romashko D., Thakur S., Mentes A., Hopkins J.F., Frampton G.M., Albacker L.A., Kohlmann A., Roberts C, & Buck E. (2022). Computational and Functional Analyses of HER2 Mutations Reveal Allosteric Activation Mechanisms and Altered Pharmacologic Effects. Cancer Research, 83(9), 1531-1542.

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