Phospho-eIF4E (Ser209) Antibody [G23F17]

Application: Reactivity: Human, Mouse, Rat

Lane 1: 293T, Lane 2: 293T (Anisomycin, 25 μg/mL, 30 min), Lane 3: Hela, Lane 4: Hela (Anisomycin, 25 μg/mL, 30 min)

Experiment Essentials

WB
Recommended wet transfer conditions: 200 mA, 60 min.

Usage Information

Dilution
WB1:1000 IP1:200

Application
WB, IP

Reactivity
Human, Mouse, Rat

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
25 kDa

Positive Control	NBT-II cells; RBL-1 cells; Rat2 cells; A-10 cells; C6 cells; 293T cells (anisomycin, 25 μg/mL, 30 min); HeLa cells (anisomycin, 25 μg/mL, 30 min)
Negative Control

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: 10%. 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, 60 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 5% skim milk powder 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: 10%. 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, 60 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 5% skim milk powder 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-eIF4E (Ser209) Antibody [G23F17] detects endogenous levels of total eIF4E protein only when it is phosphorylated at Ser209.

Subcellular Location
Chromosome, Nucleus

Uniprot ID
P06730

Clone
G23F17

Synonym(s)
Eukaryotic translation initiation factor 4E; eIF-4E; EIF4E

Background
Phospho‑eIF4E (Ser209) marks a key regulatory site on the mRNA 5′ cap‑binding initiation factor 4E (eIF4E), a central node in the control of cap‑dependent translation that functions within the eIF4F complex alongside the scaffold eIF4G and the RNA helicase eIF4A. eIF4E recognizes the 7‑methylguanosine cap of 5′‑capped mRNAs, and its interaction with eIF4G stabilizes the eIF4F complex and facilitates ribosome recruitment, while post‑translational modification at Ser209 by the MAPK‑activated kinases Mnk1 and Mnk2 fine‑tunes both the affinity and functional output of eIF4E without disrupting eIF4E–eIF4G binding per se. Activation of ERK and p38 MAPK cascades mobilizes Mnk1/2 to phosphorylate eIF4E at Ser209 within the C‑terminal domain, an event that enhances the translation of a subset of mRNAs encoding growth‑ and survival‑related proteins such as cyclins and anti‑apoptotic factors, thereby coupling extracellular cues to selective production of malignancy‑associated proteins. Ser209 phosphorylation increases resistance to oxidative, nutrient, and cytotoxic stresses by promoting recovery‑phase protein synthesis and upregulating pro‑survival factors such as Mcl‑1, and in tumorigenesis persistent eIF4E Ser209 phosphorylation drives proliferation, metastasis, and therapeutic resistance, whereas blocking Mnk‑dependent eIF4E phosphorylation suppresses eIF4E‑driven oncogenic translation and tumor growth.

Background

Phospho‑eIF4E (Ser209) marks a key regulatory site on the mRNA 5′ cap‑binding initiation factor 4E (eIF4E), a central node in the control of cap‑dependent translation that functions within the eIF4F complex alongside the scaffold eIF4G and the RNA helicase eIF4A. eIF4E recognizes the 7‑methylguanosine cap of 5′‑capped mRNAs, and its interaction with eIF4G stabilizes the eIF4F complex and facilitates ribosome recruitment, while post‑translational modification at Ser209 by the MAPK‑activated kinases Mnk1 and Mnk2 fine‑tunes both the affinity and functional output of eIF4E without disrupting eIF4E–eIF4G binding per se. Activation of ERK and p38 MAPK cascades mobilizes Mnk1/2 to phosphorylate eIF4E at Ser209 within the C‑terminal domain, an event that enhances the translation of a subset of mRNAs encoding growth‑ and survival‑related proteins such as cyclins and anti‑apoptotic factors, thereby coupling extracellular cues to selective production of malignancy‑associated proteins. Ser209 phosphorylation increases resistance to oxidative, nutrient, and cytotoxic stresses by promoting recovery‑phase protein synthesis and upregulating pro‑survival factors such as Mcl‑1, and in tumorigenesis persistent eIF4E Ser209 phosphorylation drives proliferation, metastasis, and therapeutic resistance, whereas blocking Mnk‑dependent eIF4E phosphorylation suppresses eIF4E‑driven oncogenic translation and tumor growth.

References
https://pubmed.ncbi.nlm.nih.gov/20679199/ https://pubmed.ncbi.nlm.nih.gov/25923732/

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%