Samples were denatured in buffer containing 60 mM Tris/pH 6.8, 25% glycerol, 2% sodium dodecyl sulfate (SDS), and 14.4 mM 2-mercaptoethanol with 0.1% bromophenol blue, and boiled for 5 minutes. SDS polyacrylamide gels (Bio-Rad, Hercules, CA, USA) were loaded with 25 µg of total protein per lane. Prestained molecular weight markers (Bio-Rad) were used as standards. Electrophoresed samples were transferred to polyvinylidene difluoride membranes (GE Healthcare, Piscataway, NJ, USA). After transfer, membranes were blocked with 3% bovine serum albumin in Tris-phosphate-buffered saline (TPBS; 200 mM Tris/pH 7.0, 1.37 M NaCl, 1% Tween-20) for 1 hour at room temperature. Membranes were then incubated with anti-RET, anti-phospho-RET, anti-EGFR, anti-phospho-EGFR, anti-AKT, anti-phospho-AKT, anti-ERK, anti-phospho-ERK, or anti-beta actin (1:1000; Cell Signaling Technology, Danvers, MA, USA) overnight at 4℃, washed as before, and incubated with goat anti-rabbit peroxidase-conjugated secondary antibody (1:4000; Cell Signaling Technology) for 2 hours at room temperature. Membranes were then washed, and expressed proteins were detected with Pierce ECL plus western blotting substrate (Pierce, Rockford, IL, USA). Experiments were conducted at least three times independently.
Anti phospho ret
Manufactured by Cell Signaling Technology
Sourced in United States
Anti-phospho-RET is a primary antibody that specifically detects phosphorylated forms of the RET receptor tyrosine kinase. RET is a crucial mediator of cell signaling pathways involved in various biological processes.
Automatically generated - may contain errors
Lab products found in correlation
Prestained molecular weight markers, by Bio-Rad (1 mentions)
Sds polyacrylamide gel, by Bio-Rad (1 mentions)
Anti phospho egfr, by Cell Signaling Technology (1 mentions)
Goat anti rabbit peroxidase conjugated secondary antibody, by Cell Signaling Technology (1 mentions)
Polyvinylidene difluoride membrane, by GE Healthcare (1 mentions)
Anti akt, by Cell Signaling Technology (1 mentions)
Anti erk, by Cell Signaling Technology (1 mentions)
Anti phospho erk, by Cell Signaling Technology (1 mentions)
Anti phospho akt, by Cell Signaling Technology (1 mentions)
Anti egfr, by Cell Signaling Technology (1 mentions)
Anti β actin, by Cell Signaling Technology (1 mentions)
Anti phospho erk1 2, by Cell Signaling Technology (1 mentions)
Anti erk1 2, by Cell Signaling Technology (1 mentions)
Clarity western ecl substrate, by Bio-Rad (1 mentions)
Bradford assay, by Bio-Rad (1 mentions)
2 protocols using anti phospho ret
1
Western Blot Analysis of Protein Signaling
2
Detecting RET Dimer Formation
Check if the same lab product or an alternative is used in the 5 most similar protocols
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Protein extracts were prepared with lysis buffer containing 50 mM HEPES pH 7.5, 5 mM EDTA, 250 mM NaCl, 1 mM dithiothreitol, 0.5% Nonidet P40, 1 mM Na3VO4, 1 mM NaF supplemented with 10 μg of aprotinin/ml, 10 μg of leupeptin/ml, 1 mM PMSF and a mix of protease inhibitors (SIGMAFAST protease inhibitor Tablets for general Use; Sigma-Aldrich St. Louis, MO, USA). Lysates were centrifuged at 13,000 rpm for 30 min at 4 °C and the supernatants were collected. Protein concentration was estimated with a modified Bradford assay (Bio-Rad Laboratories, Berkeley, CA, USA). Western blot analysis was carried out by standard methods.
To detect RET dimers, electrophoresis was carried out on a 6% SDS–PAGE in non-reducing conditions. In this case proteins extracts were prepared in the same lysis buffer as above devoid of dithiothreitol, and with sample loading buffer devoid of 2-mercaptoethanol. Samples were not heated upon loading.
Proteins were revealed by enhanced chemiluminescence detection using Clarity™ Western ECL Substrate (Bio-Rad Laboratories, Berkeley, CA, USA). The antibodies used in this study were: anti-RET (E1N8X, #14556), anti-phospho-RET, (#3221), anti-phospho-ERK1/2 (#9101), anti-ERK1/2 (#9107) purchased from Cell Signaling Technology, Danver, MA, USA. The anti-phospho-RET Y1062 used in this study has been previously described [27 (link)].
To detect RET dimers, electrophoresis was carried out on a 6% SDS–PAGE in non-reducing conditions. In this case proteins extracts were prepared in the same lysis buffer as above devoid of dithiothreitol, and with sample loading buffer devoid of 2-mercaptoethanol. Samples were not heated upon loading.
Proteins were revealed by enhanced chemiluminescence detection using Clarity™ Western ECL Substrate (Bio-Rad Laboratories, Berkeley, CA, USA). The antibodies used in this study were: anti-RET (E1N8X, #14556), anti-phospho-RET, (#3221), anti-phospho-ERK1/2 (#9101), anti-ERK1/2 (#9107) purchased from Cell Signaling Technology, Danver, MA, USA. The anti-phospho-RET Y1062 used in this study has been previously described [27 (link)].
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