- 711842 - Invitrogen Antibodies EEF2 antibody

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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Knockdown of EEF2 was achieved by transfecting HeLa cells with EEF2 specific siRNA (Silencer® select Product # s4493 and s4492). Western blot analysis (Fig a) was performed using whole cell extract from the EEF2 knock down cells (lane 3), non-specific scrambled siRNA transfected cells (lane 2) and untransfected cells (lane 1). The blots were probed with Anti-EEF2 Recombinant Rabbit Polyclonal Antibody (Product # 711842, 1-3 µg/mL) and Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, HRP conjugate (Product # A27036, 0.4 µg/mL, 1:2500 dilution). Densitometric analysis of this Western blot is shown in histogram (Fig b). Loss of signal upon siRNA mediated knock down confirms that antibody is specific to EEF2.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Western blot analysis was performed on Whole cell extracts (30 µg lysate) of HCT 116 (Lane 1), Hep G2 (Lane 2), HT-29 (Lane 3), HeLa (Lane 4), SH-SY5Y (Lane 5), MCF-7 (Lane 6), U-2 OS (Lane 7), A549 (Lane 8) and T-47D (Lane 9). The blots were probed with Anti-EEF2 Recombinant Rabbit Polyclonal Antibody (Product # 711842, 2.5 µg/mL) and detected by chemiluminescence using Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, HRP conjugate (Product # A27036, 0.4 µg/mL, 1:4000 dilution). A 95 kDa band corresponding to EEF2 was observed across the cell lines tested. Known quantity of protein samples were electrophoresed using Novex®NuPAGE®4-12% Bis-Tris gel (Product # NP0322BOX), 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)
Main image
Experimental details: Western blot analysis was performed on Whole cell extracts (30 µg lysate) of HCT 116 (Lane 1), Hep G2 (Lane 2), HT-29 (Lane 3), HeLa (Lane 4), SH-SY5Y (Lane 5), MCF-7 (Lane 6), U-2 OS (Lane 7), A549 (Lane 8) and T-47D (Lane 9). The blots were probed with Anti-EEF2 Recombinant Rabbit Polyclonal Antibody (Product # 711842, 2.5 µg/mL) and detected by chemiluminescence using Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, HRP conjugate (Product # A27036, 0.4 µg/mL, 1:4000 dilution). A 95 kDa band corresponding to EEF2 was observed across the cell lines tested. Known quantity of protein samples were electrophoresed using Novex®NuPAGE®4-12% Bis-Tris gel (Product # NP0322BOX), 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)
Main image
Experimental details: For immunofluorescence analysis, HCT116 cells were fixed and permeabilized for detection of endogenous EEF2 using Anti- EEF2 Recombinant Rabbit Polyclonal Antibody (Product # 711842, 5 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 EEF2 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 EEF2. Panel e) represents control cells with no primary antibody to assess background. The images were captured at 60X magnification.

711842