Phospho-EGF Receptor (Tyr998) Antibody [L7A22]

Application: Reactivity: Human

Lane 1: A431, Lane 2: A431 (EGF treated)

Experiment Essentials

WB
Recommended SDS-PAGE separating gel concentration: 5%.

Usage Information

Dilution
WB1:1000 IP1:50

Application
WB, IP

Reactivity
Human

Source
Rabbit Monoclonal Antibody

Storage Buffer
PBS, pH 7.2+50% Glycerol+0.05% BSA+0.01% NaN3

Storage (from the date of receipt)
-20°C (avoid freeze-thaw cycles), 2 years

Predicted MW
175 kDa

Positive Control	BxPC-3 cells (EGF treated); A431 cells (hEGF, 100 ng/mL, 30 min)
Negative Control	A431 cells; BxPC-3 cells

Experimental Methods

WB
Experimental Protocol: Sample preparation 1. Tissue: Lyse the tissue sample by adding an appropriate volume of ice-cold RIPA/NP-40 Lysis Buffer (containing Protease Inhibitor Cocktail, Phosphatase Inhibitor Cocktail)，and homogenize the tissue at a low temperature or lyse it by sonication on ice, then incubate on ice for 30 minutes. 2. Adherent cell: Aspirate the culture medium and wash the cells with ice-cold PBS twice. Lyse the cells by adding an appropriate volume of RIPA/NP-40 Lysis Buffer (containing Protease Inhibitor Cocktail, Phosphatase Inhibitor Cocktail) , sonicate to lyse the cells, and incubate on ice for 30 minutes. 3. Suspension cell: Transfer the culture medium to a pre-cooled centrifuge tube. Centrifuge and aspirate the supernatant. Wash the cells with ice-cold PBS twice. Lyse the cells by adding an appropriate volume of RIPA/NP-40 Lysis Buffer (containing Protease Inhibitor Cocktail, Phosphatase Inhibitor Cocktail) , sonicate to lyse the cells, and incubate on ice for 30 minutes. 4. Place the lysate into a pre-cooled microcentrifuge tube. Centrifuge at 4°C for 15 min. Collect the supernatant; 5. Remove a small volume of lysate to determine the protein concentration; 6. Combine the lysate with protein loading buffer. Boil 20 µL sample under 95-100°C for 5 min. Centrifuge for 5 min after cool down on ice. Electrophoretic separation 1. According to the concentration of extracted protein, load appropriate amount of protein sample and marker onto SDS-PAGE gels for electrophoresis. Recommended separating gel (lower gel) concentration: 5%. Reference Table for Selecting SDS-PAGE Separation Gel Concentrations 2. Power up 80V for 30 minutes. Then the power supply is adjusted (110 V~150 V), the Marker is observed, and the electrophoresis can be stopped when the indicator band of the predyed protein Marker where the protein is located is properly separated. (Note that the current should not be too large when electrophoresis, too large current (more than 150 mA) will cause the temperature to rise, affecting the result of running glue. If high currents cannot be avoided, an ice bath can be used to cool the bath.) Transfer membrane 1. Take out the converter, soak the clip and consumables in the pre-cooled converter; 2. Activate PVDF membrane with methanol for 1 min and rinse with transfer buffer; 3. Install it in the order of "black edge of clip - sponge - filter paper - filter paper - glue -PVDF membrane - filter paper - filter paper - sponge - white edge of clip"; 4. The protein was electrotransferred to PVDF membrane. ( 0.45 µm PVDF membrane is recommended ) Reference Table for Selecting PVDF Membrane Pore Size Specifications Recommended conditions for wet transfer: 200 mA, 120 min. ( Note that the transfer conditions can be adjusted according to the protein size. For high-molecular-weight proteins, a higher current and longer transfer time are recommended. However, ensure that the transfer tank remains at a low temperature to prevent gel melting.) Block 1. After electrotransfer, wash the film with TBST at room temperature for 5 minutes; 2. Incubate the film in the blocking solution ( recommending 5% BSA solution) for 1 hour at room temperature; 3. Wash the film with TBST for 3 times, 5 minutes each time. Antibody incubation 1. Use primary antibody dilution buffer to prepare the primary antibody working liquid (recommended dilution ratio for primary antibody 1:1000), gently shake and incubate with the film at 4°C overnight; 2. Wash the film with TBST 3 times, 5 minutes each time; 3. Add the secondary antibody to the blocking solution and incubate with the film gently at room temperature for 1 hour; 4. After incubation, wash the film with TBST 3 times for 5 minutes each time. Antibody staining 1. Add the prepared ECL luminescent substrate (or select other color developing substrate according to the second antibody) and mix evenly; 2. Incubate with the film for 1 minute, remove excess substrate (keep the film moist), wrap with plastic film, and expose in the imaging system.

WB

Experimental Protocol:

Sample preparation

1. Tissue: Lyse the tissue sample by adding an appropriate volume of ice-cold RIPA/NP-40 Lysis Buffer (containing Protease Inhibitor Cocktail, Phosphatase Inhibitor Cocktail)，and homogenize the tissue at a low temperature or lyse it by sonication on ice, then incubate on ice for 30 minutes.

2. Adherent cell: Aspirate the culture medium and wash the cells with ice-cold PBS twice. Lyse the cells by adding an appropriate volume of RIPA/NP-40 Lysis Buffer (containing Protease Inhibitor Cocktail, Phosphatase Inhibitor Cocktail) , sonicate to lyse the cells, and incubate on ice for 30 minutes.

3. Suspension cell: Transfer the culture medium to a pre-cooled centrifuge tube. Centrifuge and aspirate the supernatant. Wash the cells with ice-cold PBS twice. Lyse the cells by adding an appropriate volume of RIPA/NP-40 Lysis Buffer (containing Protease Inhibitor Cocktail, Phosphatase Inhibitor Cocktail) , sonicate to lyse the cells, and incubate on ice for 30 minutes.

4. Place the lysate into a pre-cooled microcentrifuge tube. Centrifuge at 4°C for 15 min. Collect the supernatant;

5. Remove a small volume of lysate to determine the protein concentration;

6. Combine the lysate with protein loading buffer. Boil 20 µL sample under 95-100°C for 5 min. Centrifuge for 5 min after cool down on ice.

Electrophoretic separation

1. According to the concentration of extracted protein, load appropriate amount of protein sample and marker onto SDS-PAGE gels for electrophoresis. Recommended separating gel (lower gel) concentration: 5%. Reference Table for Selecting SDS-PAGE Separation Gel Concentrations

2. Power up 80V for 30 minutes. Then the power supply is adjusted (110 V~150 V), the Marker is observed, and the electrophoresis can be stopped when the indicator band of the predyed protein Marker where the protein is located is properly separated. (Note that the current should not be too large when electrophoresis, too large current (more than 150 mA) will cause the temperature to rise, affecting the result of running glue. If high currents cannot be avoided, an ice bath can be used to cool the bath.)

Transfer membrane

1. Take out the converter, soak the clip and consumables in the pre-cooled converter;

2. Activate PVDF membrane with methanol for 1 min and rinse with transfer buffer;

3. Install it in the order of "black edge of clip - sponge - filter paper - filter paper - glue -PVDF membrane - filter paper - filter paper - sponge - white edge of clip";

4. The protein was electrotransferred to PVDF membrane. ( 0.45 µm PVDF membrane is recommended ) Reference Table for Selecting PVDF Membrane Pore Size Specifications

Recommended conditions for wet transfer: 200 mA, 120 min.

( Note that the transfer conditions can be adjusted according to the protein size. For high-molecular-weight proteins, a higher current and longer transfer time are recommended. However, ensure that the transfer tank remains at a low temperature to prevent gel melting.)

Block

1. After electrotransfer, wash the film with TBST at room temperature for 5 minutes;

2. Incubate the film in the blocking solution ( recommending 5% BSA solution) for 1 hour at room temperature;

3. Wash the film with TBST for 3 times, 5 minutes each time.

Antibody incubation

1. Use primary antibody dilution buffer to prepare the primary antibody working liquid (recommended dilution ratio for primary antibody 1:1000), gently shake and incubate with the film at 4°C overnight;

2. Wash the film with TBST 3 times, 5 minutes each time;

3. Add the secondary antibody to the blocking solution and incubate with the film gently at room temperature for 1 hour;

4. After incubation, wash the film with TBST 3 times for 5 minutes each time.

Antibody staining

1. Add the prepared ECL luminescent substrate (or select other color developing substrate according to the second antibody) and mix evenly;

2. Incubate with the film for 1 minute, remove excess substrate (keep the film moist), wrap with plastic film, and expose in the imaging system.

Datasheet & SDS

Biological Description

Specificity
Phospho-EGF Receptor (Tyr998) Antibody (Rabbit mAb) [L7A22] detects endogenous levels of total EGF Receptor protein only when it is phosphorylated at Tyr998.

Subcellular Location
Endoplasmic reticulum, Endosome, Golgi apparatus, Membrane, Nucleus, Secreted

Uniprot ID
P00533

Clone
L7A22

Synonym(s)
Epidermal growth factor receptor, Proto-oncogene c-ErbB-1, Receptor tyrosine-protein kinase erbB-1, EGFR, ERBB, ERBB1, HER1

Background
Phospho–EGF receptor (Tyr998) represents a ligand-inducible regulatory state of EGFR in which a tyrosine within the juxtamembrane/proximal cytoplasmic region is phosphorylated with delayed kinetics and participates primarily in the control of receptor endocytosis and trafficking rather than in classical Ras–ERK signal initiation. The receptor itself is a HER/ErbB-family type I transmembrane kinase with an extracellular ligand-binding ectodomain, a single-pass transmembrane helix, an intracellular tyrosine kinase domain, and a C‑terminal tail harboring multiple canonical autophosphorylation sites and serine/threonine clusters; Tyr998 lies N‑terminal to these major docking tyrosines and is phosphorylated together with nearby Ser991 as part of a distinct multisite phosphorylation cassette. Quantitative phosphoproteomics of EGF-stimulated carcinoma cells shows that phosphorylation at Tyr998 and Ser991 accumulates more slowly than at classical signaling sites such as Tyr1068 and Tyr1173, while receptors carrying the Y998F or S991A substitutions retain robust ERK activation and maintain EGF-stimulated GRB2 binding, indicating that Tyr998 is dispensable for proximal Ras–ERK signaling but contributes to a later regulatory phase. Specific requirement of Tyr998 and Ser991 for efficient clathrin-mediated endocytosis: Y998F and S991A receptors show impaired internalization despite normal kinase activation, reduced association of EGFR with the E3 ligase CBL, and decreased receptor ubiquitination, which collectively delay sorting into degradative pathways. Trafficking-defective Y998F and S991A receptors exhibit elevated phosphorylation of a downstream serine/threonine cluster at Ser1039 and Thr1041, residues controlled by p38 MAP kinase and previously implicated in stress-induced internalization and recycling, and pharmacologic inhibition of p38 reduces Ser1039/Thr1041 phosphorylation and alters receptor trafficking dynamics, pointing to a coordinated phosphorylation network in which Tyr998 and Ser991 “license” subsequent p38‑dependent modifications that fine-tune endocytosis and recycling. In this model, Tyr998 phosphorylation functions as part of a spatial and temporal code that links the early autophosphorylation pattern at classical docking sites to the later recruitment of CBL, ubiquitin machinery, and p38‑regulated trafficking factors, shaping the balance between sustained surface signaling and receptor downregulation in response to EGF.

Background

Phospho–EGF receptor (Tyr998) represents a ligand-inducible regulatory state of EGFR in which a tyrosine within the juxtamembrane/proximal cytoplasmic region is phosphorylated with delayed kinetics and participates primarily in the control of receptor endocytosis and trafficking rather than in classical Ras–ERK signal initiation. The receptor itself is a HER/ErbB-family type I transmembrane kinase with an extracellular ligand-binding ectodomain, a single-pass transmembrane helix, an intracellular tyrosine kinase domain, and a C‑terminal tail harboring multiple canonical autophosphorylation sites and serine/threonine clusters; Tyr998 lies N‑terminal to these major docking tyrosines and is phosphorylated together with nearby Ser991 as part of a distinct multisite phosphorylation cassette. Quantitative phosphoproteomics of EGF-stimulated carcinoma cells shows that phosphorylation at Tyr998 and Ser991 accumulates more slowly than at classical signaling sites such as Tyr1068 and Tyr1173, while receptors carrying the Y998F or S991A substitutions retain robust ERK activation and maintain EGF-stimulated GRB2 binding, indicating that Tyr998 is dispensable for proximal Ras–ERK signaling but contributes to a later regulatory phase. Specific requirement of Tyr998 and Ser991 for efficient clathrin-mediated endocytosis: Y998F and S991A receptors show impaired internalization despite normal kinase activation, reduced association of EGFR with the E3 ligase CBL, and decreased receptor ubiquitination, which collectively delay sorting into degradative pathways. Trafficking-defective Y998F and S991A receptors exhibit elevated phosphorylation of a downstream serine/threonine cluster at Ser1039 and Thr1041, residues controlled by p38 MAP kinase and previously implicated in stress-induced internalization and recycling, and pharmacologic inhibition of p38 reduces Ser1039/Thr1041 phosphorylation and alters receptor trafficking dynamics, pointing to a coordinated phosphorylation network in which Tyr998 and Ser991 “license” subsequent p38‑dependent modifications that fine-tune endocytosis and recycling. In this model, Tyr998 phosphorylation functions as part of a spatial and temporal code that links the early autophosphorylation pattern at classical docking sites to the later recruitment of CBL, ubiquitin machinery, and p38‑regulated trafficking factors, shaping the balance between sustained surface signaling and receptor downregulation in response to EGF.

References
https://pubmed.ncbi.nlm.nih.gov/19531499/ https://pubmed.ncbi.nlm.nih.gov/15657067/

Protein Size (kDa)	PVDF Transfer Membrane Pore Size (μm)
< 10	0.22
10-20	0.22
20-30	0.45
30-45	0.45
45-70	0.45
80-100	0.45
100-140	0.45
> 140	0.45

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Protein Size (kDa)	Separating Gel Percentage
4-40	20%
12-45	15%
10-70	12.5%
15-100	10%
25-100	8%
60-210	5%

Phospho-EGF Receptor (Tyr998) Antibody (Rabbit mAb) [L7A22]