Figure 1: EGFR target protein structure.

EGFR Target Introduction

Protein Function

• EGFR is one of the members of the epidermal growth factor receptor (HER) family. This family includes HER1 (erbB1, EGFR), HER2 (erbB2, NEU), HER3 (erbB3), and HER4 (erbB4).
• EGFR is a receptor for epidermal growth factor (EGF) that is involved in cell proliferation and signal transduction. It activates at least four major downstream signaling cascades, including RAS-RAF-MEK-ERK, PI3 kinase-AKT, PLCgamma-PKC, and STATs, and may also activate the NF-kappa-B signaling cascade.
• Studies have shown that EGFR is overexpressed or aberrantly expressed in many solid tumors. EGFR is associated with tumor cell proliferation, angiogenesis, tumor invasion, metastasis, and inhibition of cell apoptosis.

Protein Expression

• Widely expressed, but the expression levels vary significantly in different samples.
• Isoform 2 is also expressed in ovarian cancer.

Protein Localization

• Localized to the cell membrane, Golgi membrane, endoplasmic reticulum membrane, nuclear membrane, etc.
• Isoform 2 is a secreted protein.

Figure 2: EGFR ICC experimental result image, Anti-EGFR antibody [EP38Y] (ab52894). Green: EGFR; Red: alpha Tubulin; Blue: DAPI.

Isoforms & post-translational modifications

• Human (P00533): Isoform 1 (p170): 134 kDa (predicted), Isoform 2 (p60): 44 kDa (predicted), Isoform 3 (p110): 77 kDa (predicted), Isoform 4: 69 kDa (predicted)
• Mouse (Q01279): 134 kDa (predicted)
• Existence of phosphorylation, disulfide bonds, glycosylation, palmitoylation, methylation, lipidation, and other post-translational modifications.
• Tyrosine residue phosphorylation in response to EGF.
• Phosphorylation at Ser-695 site is partial and only occurs when Thr-693 site is phosphorylated. Phosphorylation of PRKD1 at Thr-678 and Thr-693 sites can inhibit EGF-induced activation of MAPK8/JNK1.

WB Experiment Tips

Precautions

• EGFR has expression specificity, please confirm the expression of target protein in the test sample in advance; for cell lines and tissues with low endogenous EGFR expression levels, it may be necessary to optimize experimental conditions, such as trying to concentrate the lysate to enrich the target protein, and it is also recommended to use positive controls.
• The predicted molecular weight of EGFR is 134 kDa. Due to the presence of multiple post-translational modification sites and differences in the degree of modification in different samples, the observed molecular weight may range from 170-180 kDa.
• EGFR protein has multiple phosphorylation modification sites, please select the appropriate antibody based on the exact sites of phosphorylation modification induced after stimulation in the literature.
• When detecting phosphorylation-modified EGFR, please first confirm the content of total EGFR protein in the cells. At the same time, stimulation induction can increase the content of p-EGFR protein in the cells (e.g., using EGF to stimulate A431 cells). It is difficult to detect signals in uninduced cells. Please refer to the product manual or relevant literature for specific induction conditions.

Positive controls

• EGFR: A431 whole cell lysate
• EGFR (phospho Y1068): A431 whole cell lysate stimulated with 100 ng/ml EGF for 30 minutes

Example of results

Figure 3: WB-Anti-EGFR antibody [EP38Y] (ab52894).

Lane 1: 20 µg Caco-2 whole cell lysate
Lane 2: 20 µg A431 whole cell lysate
Lane 3: 20 µg mouse skin cell whole cell lysate
Lane 4: 20 µg rat skin cell whole cell lysate

Predicted band size: 134 kDa

Figure 4: WB- Anti-EGFR (phospho Y1068) antibody [Y38] (ab32430).

Lane 1: 10 µg A431 whole cell lysate
Lane 2: 10 µg A431 whole cell lysate treated with 100 ng/ml EGF for 30 minutes
Lane 3: 10 µg A431 whole cell lysate treated with 100 ng/ml EGF for 30 minutes, followed by incubation with phosphatase

Predicted band size: 135 kDa
Actual band size: 175 kDa

Key control points

In the experiment, special attention should be given to key control points in addition to routine issues:

Sample preparation:

1. Add a complex proteinase inhibitor to avoid degradation of the target protein.
2. Keep the sample on ice throughout the sample preparation process.
3. Determine the total protein concentration of the sample by Bradford analysis, Lowry analysis, or BCA analysis.

Electrophoresis:

1. For target proteins with larger molecular weight (such as molecular weight >100 kDa), use an 8% separation gel for electrophoresis.
2. Load at least 20 μg total protein for electrophoresis.

Transfer:

1. For target proteins with larger molecular weight, we recommend adding SDS to a final concentration of 0.1% in the transfer buffer.
2. For target proteins with larger molecular weight, it is recommended to use a 0.45 μm PVDF membrane.
3. For target proteins with larger molecular weight, it is recommended to use 10% methanol or lower concentration in the transfer buffer.
4. It is recommended to use Ponceau S staining after transfer to confirm the success of the transfer.

References

1. Kristin B Runkle, Akriti Kharbanda, Ewa Stypulkowski, Xing-Jun Cao, Wei Wang, Benjamin A Garcia, Eric S Witze. Inhibition of DHHC20-Mediated EGFR Palmitoylation Creates a Dependence on EGFR Signaling. Mol Cell. 2016 May 5;62(3):385-396. doi: 10.1016/j.molcel.2016.04.003.
2. A A Habib, S Chatterjee, S K Park, R R Ratan, S Lefebvre, T Vartanian. The epidermal growth factor receptor engages receptor interacting protein and nuclear factor-kappa B (NF-kappa B)-inducing kinase to activate NF-kappa B. Identification of a novel receptor-tyrosine kinase signalosome. J Biol Chem. 2001 Mar 23;276(12):8865-74. doi: 10.1074/jbc.M008458200. Epub 2000 Dec 14.
3. Pradipta Ghosh, Anthony O Beas, Scott J Bornheimer, etc. A G{alpha}i-GIV molecular complex binds epidermal growth factor receptor and determines whether cells migrate or proliferate. Mol Biol Cell. 2010 Jul 1;21(13):2338-54. doi: 10.1091/mbc.e10-01-0028. Epub 2010 May 12.