Rabbit anti phosphoegfr

Manufactured by Cell Signaling Technology

Sourced in United States

Rabbit anti-phosphoEGFR is a primary antibody that recognizes the epidermal growth factor receptor (EGFR) when phosphorylated at specific tyrosine residues. It is designed for use in applications such as Western blotting, immunoprecipitation, and immunohistochemistry to detect phosphorylated EGFR.

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3 protocols using rabbit anti phosphoegfr

EGFR Signaling Pathway Analysis

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Tetracycline-free Fetal Bovine Serum was purchased from Clontech. Dulbecco's modified Eagle's medium was purchased from Invitrogen. Rabbit anti-EGFR (EGF Receptor (D38B1) XP^® Rabbit mAb #4267) and Rabbit anti-phosphoEGFR (Phospho-EGF Receptor (Tyr1068) (D7A5) XP^® Rabbit mAb #3777; Phospho-EGF Receptor (Tyr845) (D63B4) Rabbit mAb) antibodies were from Cell Signaling, Inc. Mouse anti-EGFR (clone 13A9) was a generous gift from Genentech Inc. The anti-EGFRvIII antibody was previously described [58 (link)]. For immunoblotting detection of EHD3, we used either a mouse monoclonal antibody (Clone RR-L, Santa Cruz Biotechnologies) or our custom-made rabbit polyclonal antibody [8 (link)]. Antibodies against Cbl, phospho-Cbl, Chk, ERK, phospho-ERK, AKT, phospho-AKT, GAPDH and Actin were from Cell Signaling.

Amessou M., Ebrahim A.S., Dilly A., Joseph M., Tabolina M., Chukkapalli S., Meroueh L., Syed J.T., Liddane A., Lang S.L., Al-Katib A, & Kandouz M. (2016). Spatio-temporal regulation of EGFR signaling by the Eps15 homology domain-containing protein 3 (EHD3). Oncotarget, 7(48), 79203-79216.

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Western Blot Analysis of Liver Tissue

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Liver tissues and cell samples were homogenized in radio immunoprecipitation assay buffer (Thermo Fisher Scientific) containing protease and phosphatase inhibitor cocktail (Thermo Fisher Scientific). Protein concentrations were quantified with a commercial BCA protein assay kit (Thermo Fisher Scientific) according to the manufacturer’s instructions. At least 30 μg of total protein from each sample were loaded and separated by gel electrophoresis and then transferred to nitrocellulose membranes. After blocking, membranes were incubated with primary antibodies at 4°C overnight under shaking conditions. The membranes were then incubated with horseradish peroxidase–conjugated secondary antibodies (Jackson ImmunoResearch Laboratories). Detection and quantification of protein bands were performed using a ChemiDoc Imaging System with ImageLab Software (Bio-Rad Laboratories). The primary antibodies used were mouse anti-β-actin (Sigma-Aldrich), mouse anti–proliferating cell nuclear antigen (PCNA), rabbit anti-phospho STAT3 (Tyr705), rabbit anti-STAT3, rabbit anti-phospho EGFR (Tyr1086), rabbit anti-EGFR, rabbit anti-phospho-MET (Thy1234/1235), rabbit anti-MET, rabbit anti-phospho-AKT (Ser473), rabbit anti-AKT, rabbit anti-phospho ERK1/2 (Thr202/Tyr204), rabbit anti-EKR1/2, rabbit anti-phospho-p70S6K (Thr421/Ser424), and rabbit anti-p70S6K (Cell Signaling Technology).

Wen Y., Emontzpohl C., Xu L., Atkins C.L., Jeong J.M., Yang Y., Kim K., Wu C., Akira S, & Ju C. (2023). Interleukin-33 facilitates liver regeneration through serotonin-involved gut-liver axis. Hepatology (Baltimore, Md.), 77(5), 1580-1592.

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Protein Extraction and Western Blot Analysis

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The cells were lysed with RIPA buffer (Beyotime, Shanghai, China) containing 1X PMSF and a protease inhibitor cocktail. After centrifugation at 4 °C and 12,000× g for 15 min, the concentration of proteins in the supernatant was determined using a Piece BCA protein assay kit (Thermo Fisher Scientific Inc., Waltham, MA, USA). The proteins were separated using 10% sodium dodecyl-sulfate-polyacrylamide gels. Thereafter, they were transferred onto a PVDF membrane using a transfer apparatus, and after blocking with 5% skimmed milk, the membrane was incubated with the primary antibody overnight at 4 °C. The primary antibodies, rabbit anti-EGFR, rabbit anti-phospho-EGFR, rabbit anti-FASN, rabbit anti-LIPIN1, rabbit anti-Insig1, rabbit anti-SCD1, rabbit anti-SREBP1, rabbit anti-pAMPK, rabbit anti-p-AKT, rabbit anti-p-PI3K, and mouse anti-GAPDH, were purchased from Cell Signaling Technology (Danvers, MA, USA). Further, rabbit anti-Srebf1 was purchased from Abcam (Cambridge, MA, USA), while goat anti-rabbit IgG and goat anti-mouse secondary antibodies were purchased from Proteintech (Rosemont, IL, USA).

Liao T., Deng J., Chen W., Xu J., Yang G., Zhou M., Lv Z., Wang S., Song S., Tan X., Yin Z., Li Y, & Jin Y. (2022). Fasudil Increased the Sensitivity to Gefitinib in NSCLC by Decreasing Intracellular Lipid Accumulation. Cancers, 14(19), 4709.

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