- PA5-17745 - Invitrogen Antibodies EIF4EBP1 antibody

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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Knockout of 4EBP1 was achieved by CRISPR-Cas9 genome editing using LentiArray™ Lentiviral sgRNA (Product # A32042, Assay ID CRISPR648136_LV) and LentiArray Cas9 Lentivirus (Product # A32064). Western blot analysis of 4EBP1 was performed by loading 30 µg of HeLa Wild Type (Lane 1), HeLa Cas9 (Lane 2) andHeLa 4EBP1 KO (Lane 3) whole cell extracts. The samples were electrophoresed using NuPAGE™ Novex™ 4-12% Bis-Tris Protein Gel (Product # NP0322BOX). Resolved proteins were then transferred onto a nitrocellulose membrane (Product # IB23001) by iBlot® 2 Dry Blotting System (Product # IB21001). The blot was probed with Anti-4EBP1 Polyclonal Antibody (Product # PA5-17745, 1:1,000 dilution) and Goat anti-Rabbit IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A27036, 1:4,000 dilution) using the iBright FL 1000 (Product # A32752). Chemiluminescent detection was performed using Novex® ECL Chemiluminescent Substrate Reagent Kit (Product # WP20005). Loss of signal upon CRISPR mediated knockout (KO) using the LentiArray™ CRISPR product line confirms that antibody is specific to 4EBP1.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Knockdown of 4EBP1 was achieved by transfecting A-431 with 4EBP1 specific siRNAs (Silencer® select Product # S223471, S223472). Western blot analysis (Fig. a) was performed using Whole cell extracts from the 4EBP1 untransfected cells (lane 1), non-targeting scrambled siRNA transfected cells (lane 2) and knockdown cells (lane 3). The blot was probed with 4EBP1 Polyclonal Antibody (Product # PA5-17745, 1:1000 dilution) and Goat anti-Rabbit IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A27036, 1:4000 dilution). Densitometric analysis of this western blot is shown in histogram (Fig. b). Decrease in signal upon siRNA mediated knock down confirms that antibody is specific to 4EBP1.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Western blot was performed using Anti-4EBP1 Polyclonal Antibody(Product # PA5-17745) and a 19kDa band corresponding to 4EBP1 was observed across cell lines tested. Whole cell extracts (30 µg lysate) of HeLa (Lane 1), A-431 (Lane 2), Hep G2 (Lane 3), BeWo (Lane 4), HEK-293 (Lane 5), U-87 MG (Lane 6) were electrophoresed using NuPAGE™ 4-12% Bis-Tris Protein Gel (Product # NP0322BOX). 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 4EBP1 was performed using 70% confluent log phase HeLa cells. 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 4EBP1 Polyclonal Antibody (Product # PA5-17745) at 5 µg in 0.1% BSA, incubated at 4 degree celsius overnight and then labeled with Goat anti-Rabbit IgG (H+L) Superclonal™ Recombinant Secondary Antibody, Alexa Fluor® 488 conjugate (Product # A27034), (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 dilution). Panel d represents the merged image showing Cytoplasm localization. Panel e represents control cells with no primary antibody to assess background. The images were captured at 60x magnification.

PA5-17745