- 702466 - Invitrogen Antibodies NF2 antibody

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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Knockdown of NF2 was achieved by transfecting PC3 cells with NF2 specific siRNA (Silencer® select Product # s224112 and Product # s194647). Western blot analysis was performed using whole cell lysates from the NF2 knock down cells (lane 3), non-specific scrambled siRNA transfected cells (lane 2) and untransfected cells (lane 1). The blots were probed with Anti-NF2 Recombinant Rabbit Monoclonal Antibody (Product # 702466, 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 NF2.

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 DU 145 (Lane 1), HUVEC (Lane 2), PANC-1 (Lane 3), U-2 OS (Lane 4), A-431 (Lane 5), PC-3 (Lane 6), MCF-7 (Lane 7) and tissue extracts of Mouse Brain (Lane 8) and Rat Brain (Lane 9). The blots were probed with Anti-NF2 (Merlin) Recombinant Rabbit Monoclonal Antibody (Product # 702466, 1 µg/mL). A 70 kDa band corresponding to NF2 (Merlin) was observed across the cell lines and tissues 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, DU-145 cells were fixed and permeabilized for detection of endogenous NF2 using Anti-NF2 Recombinant Rabbit Monoclonal Antibody (Product # 702466, 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 NF2 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 NF2. 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)
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Experimental details: Flow Cytometry analysis of endogenous NF2 was performed on U-2 OS cells labeled with Anti-NF2 Recombinant Rabbit Monoclonal Antibody (Product # 702466, 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).

702466