Phospho-eIF4E (S209) Recombinant Rabbit Monoclonal Antibody [SU0396]
cat.: ET1608-66
Product Type: Recombinant Rabbit monoclonal IgG, primary antibodies
Species reactivity: Human, Mouse, Rat
Applications: WB, IF-Cell, IHC-P, IP, FC
Clonality: Monoclonal
Clone number: SU0396
Form: Liquid
Storage condition: Store at +4℃ after thawing. Aliquot store at -20℃ or -80℃. Avoid repeated freeze / thaw cycles.
Storage buffer: 1*TBS (pH7.4), 0.05% BSA, 40% Glycerol. Preservative: 0.05% Sodium Azide.
Concentration: 1ug/ul
Purification: Protein A affinity purified.
Molecular weight: Predicted band size: 25 kDa
Isotype: IgG
Immunogen: Synthetic phospho-peptide corresponding to residues surrounding Ser209 of human eIF4E.
Positive control: HeLa cell lysate, HeLa treated with 50nM dexamethasone for 16 hours cell lysate, NIH/3T3 cell lysate, Mouse spleen tissue lysate, Rat spleen tissue lysate, NIH/3T3, human pancreas tissue, mouse brain tissue, mouse hippocampus tissue, rat spleen tissue, 293T.
Subcellular location: Cytoplasm, Nucleus.
Recommended Dilutions:
  WB
  IF-Cell
  IHC-P
  IP
  FC
1:2,000
1:50-1:200
1:50-1:200
1-2μg/sample
1:1,000
Uniprot #: SwissProt: P06730 Human | P63073 Mouse | P63074 Rat
Alternative names: AUTS19 CBP eIF 4E eIF 4F 25 kDa subunit EIF 4F eIF-4E eIF-4F 25 kDa subunit eIF4E EIF4E1 EIF4EL1 EIF4F Eukaryotic translation initiation factor 4 E Eukaryotic translation initiation factor 4E Eukaryotic translation initiation factor 4E like 1 IF4E_HUMAN Messanger RNA Cap Binding Protein eIF 4E MGC111573 mRNA cap binding protein mRNA cap-binding protein
Images
ET1608-66_1.jpg Fig1: Western blot analysis of Phospho-eIF4E (S209) on different lysates with Rabbit anti-Phospho-eIF4E (S209) antibody (ET1608-66) at 1/2,000 dilution.

Lane 1: HeLa cell lysate
Lane 2: HeLa treated with 50nM dexamethasone for 16 hours cell lysate
Lane 3: NIH/3T3 cell lysate
Lane 4: Mouse spleen tissue lysate
Lane 5: Rat spleen tissue lysate
Lane 6: HeLa treated with 50nM dexamethasone for 16 hours cell lysate, then the membrane treated with λpp for 1 hour

Lysates/proteins at 20 µg/Lane.

Predicted band size: 25 kDa
Observed band size: 25 kDa

Exposure time: 42 seconds; ECL: K1801;

4-20% SDS-PAGE gel.

Proteins were transferred to a PVDF membrane and blocked with 5% NFDM/TBST for 1 hour at room temperature. The primary antibody (ET1608-66) at 1/2,000 dilution was used in 5% NFDM/TBST at 4℃ overnight. Goat Anti-Rabbit IgG - HRP Secondary Antibody (HA1001) at 1/50,000 dilution was used for 1 hour at room temperature.
ET1608-66_2.jpg Fig2: Immunocytochemistry analysis of NIH/3T3 cells labeling Phospho-eIF4E (S209) with Rabbit anti-Phospho-eIF4E (S209) antibody (ET1608-66) at 1/100 dilution.

Cells were fixed in 4% paraformaldehyde for 15 minutes at room temperature, permeabilized with 0.1% Triton X-100 in PBS for 15 minutes at room temperature, then blocked with 1% BSA in 10% negative goat serum for 1 hour at room temperature. Cells were then incubated with Rabbit anti-Phospho-eIF4E (S209) antibody (ET1608-66) at 1/100 dilution in 1% BSA in PBST overnight at 4 ℃. Goat Anti-Rabbit IgG H&L (iFluor™ 488, HA1121) was used as the secondary antibody at 1/1,000 dilution. PBS instead of the primary antibody was used as the secondary antibody only control. Nuclear DNA was labelled in blue with DAPI.

Beta tubulin (HA601187, red) was stained at 1/100 dilution overnight at +4℃. Goat Anti-Mouse IgG H&L (iFluor™ 594, HA1126) was used as the secondary antibody at 1/1,000 dilution.
ET1608-66_3.jpg Fig3: Immunohistochemical analysis of paraffin-embedded human pancreas tissue with Rabbit anti-Phospho-eIF4E (S209) antibody (ET1608-66) at 1/200 dilution.

The section was pre-treated using heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0) for 20 minutes. The tissues were blocked in 1% BSA for 20 minutes at room temperature, washed with ddH2O and PBS, and then probed with the primary antibody (ET1608-66) at 1/200 dilution for 1 hour at room temperature. The detection was performed using an HRP conjugated compact polymer system. DAB was used as the chromogen. Tissues were counterstained with hematoxylin and mounted with DPX.
ET1608-66_4.jpg Fig4: Immunohistochemical analysis of paraffin-embedded mouse brain tissue with Rabbit anti-Phospho-eIF4E (S209) antibody (ET1608-66) at 1/200 dilution.

The section was pre-treated using heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0) for 20 minutes. The tissues were blocked in 1% BSA for 20 minutes at room temperature, washed with ddH2O and PBS, and then probed with the primary antibody (ET1608-66) at 1/200 dilution for 1 hour at room temperature. The detection was performed using an HRP conjugated compact polymer system. DAB was used as the chromogen. Tissues were counterstained with hematoxylin and mounted with DPX.
ET1608-66_5.jpg Fig5: Immunohistochemical analysis of paraffin-embedded mouse hippocampus tissue with Rabbit anti-Phospho-eIF4E (S209) antibody (ET1608-66) at 1/200 dilution.

The section was pre-treated using heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0) for 20 minutes. The tissues were blocked in 1% BSA for 20 minutes at room temperature, washed with ddH2O and PBS, and then probed with the primary antibody (ET1608-66) at 1/200 dilution for 1 hour at room temperature. The detection was performed using an HRP conjugated compact polymer system. DAB was used as the chromogen. Tissues were counterstained with hematoxylin and mounted with DPX.
ET1608-66_6.jpg Fig6: Immunohistochemical analysis of paraffin-embedded rat spleen tissue using anti-Phospho-eIF4E (S209) antibody. The section was pre-treated using heat mediated antigen retrieval with Tris-EDTA buffer (pH 8.0-8.4) for 20 minutes.The tissues were blocked in 5% BSA for 30 minutes at room temperature, washed with ddH2O and PBS, and then probed with the primary antibody (ET1608-66, 1/50) for 30 minutes at room temperature. The detection was performed using an HRP conjugated compact polymer system. DAB was used as the chromogen. Tissues were counterstained with hematoxylin and mounted with DPX.
ET1608-66_7.jpg Fig7: Phospho-eIF4E (S209) was immunoprecipitated from 0.2 mg NIH/3T3 cell lysate with ET1608-66 at 2 µg/10 µl beads. Western blot was performed from the immunoprecipitate using ET1608-66 at 1/2,000 dilution. Anti-Rabbit IgG for IP Nano-secondary antibody (NBI01H) at 1/5,000 dilution was used for 1 hour at room temperature.

Lane 1: NIH/3T3 cell lysate (input)
Lane 2: ET1608-66 IP in NIH/3T3 cell lysate
Lane 3: Rabbit IgG instead of ET1608-66 in NIH/3T3 cell lysate

Blocking/Dilution buffer: 5% NFDM/TBST
Exposure time: 15 seconds; ECL: K1801
ET1608-66_8.jpg Fig8: Flow cytometric analysis of 293T cells labeling Phospho-eIF4E (S209).

Cells were fixed and permeabilized. Then stained with the primary antibody (ET1608-66, 1/1,000) (red) compared with Rabbit IgG Isotype Control (green). After incubation of the primary antibody at +4℃ for an hour, the cells were stained with a iFluor™ 488 conjugate-Goat anti-Rabbit IgG Secondary antibody (HA1121) at 1/1,000 dilution for 30 minutes at +4℃. Unlabelled sample was used as a control (cells without incubation with primary antibody; black).
Note: All products are “FOR RESEARCH USE ONLY AND ARE NOT INTENDED FOR DIAGNOSTIC OR THERAPEUTIC USE”.