Immunoblotting was conducted as previously reported30 (link). Briefly, cells were washed twice in ice-cold PBS and lysed in lysis buffer [50 mM Tris (pH 7.4), 100 mM NaCl, 0.1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 1 mM Na3VO4, 1× protease inhibitor (Roche) and PhosSTOP (Roche)]. Whole cell extracts were separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and electro-transferred onto polyvinylidene difluoride membranes. Membranes were probed with the following primary antibodies: anti-RUNX1 (sc-365644, Santa Cruz Biotechnology, Inc.), anti-phosphorylated HER2 (#2243, Cell Signaling Technology), anti-HER2 (#2165, Cell Signaling Technology), anti-SOS1 (sc-10803, Santa Cruz Biotechnology, Inc.), anti-GAPDH (sc-47724, Santa Cruz Biotechnology, Inc.). For secondary antibodies, HRP-conjugated anti-rabbit IgG (#7074, Cell Signaling Technology) and anti-mouse IgG (#7076, Cell Signaling Technology) were used. Blots were visualized using Chemi-Lumi One Super (nacalai tesque, Inc.) and ChemiDocTM XRS + Imager (Bio-Rad Laboratories, Inc.) according to the manufacturer’s recommendations. We used total protein from human adult normal tissue (#P1234248, Biochain Inst., Inc.) as a normal control of stomach in Fig. 2c .
Anti phosphorylated her2
Manufactured by Cell Signaling Technology
Sourced in United States
Anti-phosphorylated HER2 is a laboratory reagent designed to detect the phosphorylated form of the human epidermal growth factor receptor 2 (HER2) protein. It is used in various research applications to study signaling pathways and protein modifications related to HER2 activation.
Automatically generated - may contain errors
Lab products found in correlation
Anti her2, by Cell Signaling Technology (3 mentions)
Hrp conjugated anti rabbit igg, by Cell Signaling Technology (2 mentions)
Anti akt, by Cell Signaling Technology (2 mentions)
Anti phosphorylated akt, by Cell Signaling Technology (2 mentions)
Anti gapdh, by Santa Cruz Biotechnology (1 mentions)
Chemi lumi one super, by Nacalai Tesque (1 mentions)
Chemidoc xrs imager, by Bio-Rad (1 mentions)
Anti runx1, by Santa Cruz Biotechnology (1 mentions)
Anti sos1, by Santa Cruz Biotechnology (1 mentions)
Protease inhibitor, by Roche (1 mentions)
Phosstop, by Roche (1 mentions)
Phosphatase inhibitor cocktail, by Beyotime (1 mentions)
Imagequant las 4000 biomolecular imager, by GE Healthcare (1 mentions)
Anti actin, by Santa Cruz Biotechnology (1 mentions)
Protease inhibitor cocktail, by Beyotime (1 mentions)
Bca assay kit, by Beyotime (1 mentions)
Chemiluminescence detection system, by PerkinElmer (1 mentions)
Anti phosphorylated erk1 2, by Cell Signaling Technology (1 mentions)
Anti phosphorylated egfr, by Cell Signaling Technology (1 mentions)
Las 3000 instrument, by Fujifilm (1 mentions)
3 protocols using anti phosphorylated her2
1
Immunoblotting Procedure for Protein Analysis
2
Western Blot Analysis of Signaling Proteins
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Cells were lysed using RIPA buffer supplemented with a phosphatase inhibitor cocktail and a protease inhibitor cocktail (Beyotime, Shanghai, China), and cell lysates were quantified using the BCA assay kit (Beyotime, Shanghai, China). Electrophoresis was performed to separate the proteins which were then transferred to PVDF membrane. The membranes were blocked with 5% BSA/TBST and probed with the following primary antibodies: anti-phosphorylated HER2 (#2243, Cell Signaling Technology, Beverly, MA, USA), anti-HER2 (#2165, Cell Signaling Technology), anti-Akt (#4691, Cell Signaling Technology), anti-phosphorylated Akt (#4060, Cell Signaling Technology), anti-ITGβ5 (sc-14010, Santa Cruz), anti-actin (sc-58673, Santa Cruz). After being washed by TBST for 3 times, the membranes were then incubated with secondary antibodies: HRP-conjugated anti-rabbit IgG (#7074, Cell Signaling Technology) or anti-mouse IgG (#7076, Cell Signaling Technology). After the unbound secondary antibodies were washed, the membrane was developed with Super ECL Detection Reagent (Yeason Biotechnology, Shanghai, China), and images were captured using ImageQuant™ LAS 4000 biomolecular imager (GE Healthcare).
3
Comprehensive Immunoblot Analysis of HER Family Signaling
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Immunoblot analysis was performed as previously described(16 (link)) with primary antibodies as follows: anti-HER2 (#2242), anti-phosphorylated HER2 (#2249), anti-Akt (#9272), anti-phosphorylated Akt (#4060), anti-Erk1/2 (#9102), anti-phosphorylated Erk1/2 (#4370), anti-EGFR (#4267), anti-phosphorylated EGFR (#3777), anti-phosphorylated HER3 (#4791) (all of which were obtained from Cell Signaling Technology, Danvers, MA, USA), anti-HER3 (M7297, DAKO, Glostrup, Denmark) and anti-α-tubulin (Sigma-Aldrich, St. Louis, MO, USA). Immune complexes were detected with HRP-conjugated secondary antibodies, a chemiluminescence detection system (Perkin-Elmer, Waltham, MA, USA) and an LAS-3000 instrument (Fujifilm, Tokyo, Japan).
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