- 711505 - Invitrogen Antibodies EIF4A1 antibody

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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Knockdown of E1F4A1 was achieved by transfecting HeLa cells with EIF4A1 specific siRNA (Silencer® select Product # s4567). Western blot analysis was performed using TEB extract from the EIF4A1 knock down cells (lane 3), non-specific scrambled siRNA transfected cells (lane 2) and untransfected cells (lane 1). The blots were probed with Anti-EIF4A1 Recombinant Rabbit Polyclonal Antibody (Product # 711505, 1-2 µg/mL) and Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, HRP conjugate (Product # A27036, 0.4 µg/mL, 1:2500 dilution). Reduction of signal upon siRNA mediated knock down confirms that antibody is specific to EIF4A1.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot analysis was performed on Whole cell extracts (30 µg lysate) of HeLa (Lane 1), MCF7 (Lane 2), COS-7 (Lane 3), HEK-293 (Lane 4), C2C12 (Lane 5), Hep G2 (Lane 6), PANC-1 (Lane 7), PC-3 (Lane 8) and Caco-2 (Lane 9). The blots were probed with Anti-eIF4A1 Recombinant Rabbit Polyclonal Antibody (Product # 711505, 1-2 µg/mL). A 46 kDa band corresponding to eIF4A1 was observed across the cell lines tested. The blots were detected by chemiluminescence using Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, HRP conjugate (Product # A27036, 0.4 µg/mL, 1:5000 dilution). Known quantity of protein samples were electrophoresed using Novex® NuPAGE® 4-12% Bis-Tris gel (Product # NP0321BOX), XCell SureLock™ Electrophoresis System (Product # EI0002) and Novex® Sharp Pre-Stained Protein Standard (Product # LC5800). Resolved proteins were then transferred onto a nitrocellulose membrane with iBlot® Dry Blotting System (Product # IB21001). The membrane was probed with the relevant primary and secondary Antibody following blocking with 5% skimmed milk. Chemiluminescent detection was performed using Pierce™ ECL Western blotting Substrate (Product # 32106).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: For immunofluorescence analysis, MCF7 cells were fixed and permeabilized for detection of endogenous EIF4A1 using Anti- EIF4A1 Recombinant Rabbit Polyclonal Antibody (Product # 711505, 2 µg/mL) and labeled with Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, Alexa Fluor® 488 conjugate (Product # A27034, 1:2000). Panel a) shows representative cells that were stained for detection and localization of EIF4A1 protein (green), Panel b) is stained for nuclei (blue) using SlowFade® Gold Antifade Mountant with DAPI (Product # S36938). Panel c) represents cytoskeletal F-actin staining using Rhodamine Phalloidin (Product # R415, 1:300). Panel d) is a composite image of Panels a, b and c clearly demonstrating cytoplasmic localization of EIF4A1. Panel e) represents control cells with no primary antibody to assess background. The images were captured at 60X magnification.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Knockdown of eIF4A1 was achieved by transfecting HeLa cells with eIF4A1 specific siRNA (Silencer® select Product # s4567). Flow cytometry analysis was performed on cells transfected with eIF4A1 specific siRNA, non-specific scrambled siRNA and untransfected cells. Cells were probed with Anti-eIF4A1 Recombinant Rabbit Polyclonal Antibody (Product # 711505, 5 µg/ 1M cells) or with the matched rabbit isotype control, and detected with Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody (Alexa Fluor® 488 conjugate, Product # A27034, 0.4 µg/mL, 1:2500). A representative of 10,000 cells were acquired and analyzed for each sample using an Attune® Acoustic Focusing Cytometer (Product # 4468770). No positive events were observed in siRNA transfected cells confirming specificity of the antibody to eIF4A1.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Knockdown of eIF4A1 was achieved by transfecting HeLa cells with eIF4A1 specific siRNA (Silencer® select Product # s4567). Flow cytometry analysis was performed on cells transfected with eIF4A1 specific siRNA, non-specific scrambled siRNA and untransfected cells. Cells were probed with Anti-eIF4A1 Recombinant Rabbit Polyclonal Antibody (Product # 711505, 5 µg/ 1M cells) or with the matched rabbit isotype control, and detected with Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody (Alexa Fluor® 488 conjugate, Product # A27034, 0.4 µg/mL, 1:2500). A representative of 10,000 cells were acquired and analyzed for each sample using an Attune® Acoustic Focusing Cytometer (Product # 4468770). No positive events were observed in siRNA transfected cells confirming specificity of the antibody to eIF4A1.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Flow Cytometry analysis of endogenous eIF4A1 was performed on HeLa cells labeled with Anti-eIF4A1 Recombinant Rabbit Polyclonal Antibody (Product # 711505, 5 µg/ 1M cells) or with rabbit isotype control, and detected with Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, (Alexa Fluor® 488 conjugate, Product # A27034, 0.4 µg/mL, 1:2500). A representative of 10,000 cells were acquired and analyzed for each sample using an Attune® Acoustic Focusing Cytometer (Product # 4468770).

711505