Antibody data
- Antibody Data
- Antigen structure
- References [0]
- Comments [0]
- Validations
- Western blot [1]
- Immunocytochemistry [1]
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Validation data
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- Product number
- PA5-17919 - Provider product page
- Provider
- Invitrogen Antibodies
- Product name
- Phospho-eIF4E (Ser209) Polyclonal Antibody
- Antibody type
- Polyclonal
- Antigen
- Synthetic peptide
- Description
- It is not recommended to aliquot this antibody.
- Reactivity
- Human, Mouse, Rat
- Host
- Rabbit
- Isotype
- IgG
- Vial size
- 100 µL
- Concentration
- 2 µg/mL
- Storage
- -20°C
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Supportive validation
- Submitted by
- Invitrogen Antibodies (provider)
- Main image
- Experimental details
- Western Blot was performed using Anti-Phospho-eIF4E (Ser209) Polyclonal Antibody (Product # PA5-17919) and a 30 kDa band corresponding to Eukaryotic translation initiation factor 4E was observed to be either induced in HEK-293 and Hep G2 or increased in HeLa and L6, upon different treatments. Whole cell extracts (30 µg lysate) of HeLa (Lane 1), HeLa serum starved (SS) for 16 hours and serum released (SR) with 20% FBS for 30 minutes (Lane 2), HEK-293 (Lane 3), HEK-293 treated with Anisomycin (25 µg/mL for 30 minutes) (Lane 4), L6 (Lane 5), L6 treated with Insulin (200 ng/mL for 10 minutes) (Lane 6), Hep G2 (Lane 7) and Hep G2 treated with Calyculin A (100 nm for 60 minutes) (Lane 8) were electrophoresed using NuPAGE™ 10% Bis-Tris Protein Gel (Product # NP0302BOX). Resolved proteins were then transferred onto a Nitrocellulose membrane (Product # IB23001) by iBlot® 2 Dry Blotting System (Product # IB21001). The Blot was probed with the primary antibody (1:1000 dilution) and detected by chemiluminescence with Goat anti-Rabbit IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A27036, 1:4000 dilution) using the iBright FL 1000 (Product # A32752). Chemiluminescent detection was performed using Novex® ECL Chemiluminescent Substrate Reagent Kit (Product # WP20005).
Supportive validation
- Submitted by
- Invitrogen Antibodies (provider)
- Main image
- Experimental details
- Immunofluorescence analysis of Eukaryotic translation initiation factor 4E was performed using 70% confluent log phase HeLa and HeLa cells, serum starved (SS) for 16 hours and serum released (SR) with 20% FBS for 30 minutes. The cells were fixed with 4% paraformaldehyde for 10 minutes, permeabilized with 0.1% Triton™ X-100 for 15 minutes, and blocked with 2% BSA for 45 minutes at room temperature. The cells were labeled with Phospho-eIF4E (Ser209) Polyclonal Antibody (Product # PA5-17919) at 1:100 dilution in 0.1% BSA, incubated at 4 degree celsius overnight and then labeled with Donkey anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor Plus 488 (Product # A32790), (1:2000 dilution), for 45 minutes at room temperature (Panel a: Green). Nuclei (Panel b:Blue) were stained with ProLong™ Diamond Antifade Mountant with DAPI (Product # P36962). F-actin (Panel c: Red) was stained with Rhodamine Phalloidin (Product # R415, 1:300). Panel d represents the merged image showing Cytoplasmic localization. Panel e represents untreated HeLa cells with no signal. Panel f represents control cells with no primary antibody to assess background. The images were captured at 60X magnification.