- 702241 - Invitrogen Antibodies YWHAE antibody

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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot analysis was performed on tissue extracts (30 µg lysate) of Mouse Brain (Lane 1), Rat Brain (Lane 2) and Membrane enriched cell extracts of Jurkat (Lane 3), PC12 (Lane 4), SW480 (Lane 5), NIH/3T3 (Lane 6), HepG2 (Lane 7), HeLa (Lane 8) and T-47D (Lane 9). The blots were probed with Anti-14-3-3 protein epsilon/YWHAE Recombinant Rabbit Monoclonal Antibody (Product # 702241, 1-2 µg/mL) and detected by chemiluminescence using Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, HRP conjugate (Product # A27036, 0.4 µg/mL, 1:2500 dilution). A 29 kDa band corresponding to YWHAE was observed across the tissues and cell lines tested, * non specific bands are also observed. 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: Knockdown of 14-3-3 epsilon was achieved by transfecting SH-SY5Y with 14-3-3 epsilon specific siRNAs (Silencer® select Product # s200463). Western blot analysis (Fig. a) was performed using modified whole cell extracts from the SH-SY5Y knockdown cells (lane 3), non-specific scrambled siRNA transfected cells (lane 2) and untransfected cells (lane 1). The blot was probed with 14-3-3 epsilon Antibody (5H10L5), ABfinity Rabbit Monoclonal (Product # 702241, 0.25 µg/mL ) 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 14-3-3 epsilon.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunofluorescence was performed on methanol fixed Hep G2 cells for detection of endogenous 14-3-3 protein epsilon using 14-3-3 protein epsilon/YWHAE Recombinant Rabbit Monoclonal Antibody (Product # 702241, 2µg/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 14-3-3 protein epsilon (green), Panel b) is stained for nuclei (blue) using SlowFade® Gold Antifade Mountant with DAPI (Product # S36938). Panel c) Cytoskeleton was stained with alpha-tubulin Monoclonal Antibody (Product # 32-2500, 1µg/mL) followed by Goat anti-Mouse IgG Secondary Antibody, Alexa Fluor® 594 conjugate (Product # A-11032, 1:400). Panel d) is a composite image of Panels a, b and c clearly demonstrating cytoplasmic localisation of 14-3-3 protein epsilon/YWHAE. Panel e) represents control cells with no primary antibody to assess background. The images were captured at 60X magnification.

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