ET1608-66

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:	Mouse spleen tissue lysate, rat spleen tissue lysate, NIH3T3 cell lysate, N2A, SH-SY5Y, 293, human pancreas tissue, rat spleen tissue, mouse brain tissue, mouse hippocampus tissue.
Subcellular location:	Cytoplasm, Nucleus.
Recommended Dilutions: WB IF-Cell IHC-P IP FC	1:500-1:2,000 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

	Fig1: Western blot analysis of Phospho-eIF4E (S209) on different lysates. Proteins were transferred to a PVDF membrane and blocked with 5% BSA in PBS for 1 hour at room temperature. The primary antibody (ET1608-66, 1/500) was used in 5% BSA at room temperature for 2 hours. Goat Anti-Rabbit IgG - HRP Secondary Antibody (HA1001) at 1:5,000 dilution was used for 1 hour at room temperature. Positive control: Lane 1: mouse spleen tissue lysate Lane 2: rat spleen tissue lysate
	Fig2: Western blot analysis of Phospho-eIF4E (S209) on NIH-3T3 cell lysates. Lane 1: NIH-3T3 cells, whole cell lysate, 10 μg /lane. Lane 2: NIH-3T3 cells were treated with 2.8μg/ul lambda-PP for 30 minutes, whole cell lysates, 10 μg/lane. Proteins were transferred to a PVDF membrane and blocked with 5% BSA in PBS for 1 hour at room temperature. The primary antibody Anti-Phospho-eIF4E (S209) (ET1608-66, 1/500) and Anti-GAPDH antibody (ET1601-4, 1/10,000)was used in 5% BSA at room temperature for 2 hours. Goat Anti-Rabbit IgG H&L (HRP) Secondary Antibody (HA1001) at 1:200,000 dilution was used for 1 hour at room temperature. Predicted band size: 25 kDa Observed band size: 25 kDa Exposure time: 2 minutes 29 seconds
	Fig3: Western blot analysis of Phospho-eIF4E (S209) on rat spleen tissues lysates. Lane 1: Rat spleen tissues, whole tissue lysates, 20 μg /lane. Lane 2: Rat spleen tissues were treated with 2.8μg/ul lambda-PP for 30 minutes, whole tissue lysates, 20 μg/lane. Proteins were transferred to a PVDF membrane and blocked with 5% BSA in PBS for 1 hour at room temperature. The primary antibody Anti-Phospho-eIF4E (S209) (ET1608-66, 1/500) and Anti-GAPDH antibody (ET1601-4, 1/10,000)was used in 5% BSA at room temperature for 2 hours. Goat Anti-Rabbit IgG H&L (HRP) Secondary Antibody (HA1001) at 1:200,000 dilution was used for 1 hour at room temperature. Predicted band size: 25 kDa Observed band size: 25 kDa Exposure time: 3 seconds

	Fig4: 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.
	Fig5: 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.
	Fig6: 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.

	Fig7: 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.
	Fig8: 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.
	Fig9: 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
	Fig10: 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”.