- 710947 - Invitrogen Antibodies ACTN2 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) of Mouse skeletal muscle (Lane 1), Rat skeletal muscle (Lane 2), Mouse heart (Lane 3), Rat Heart (Lane 4), Mouse Brain (Lane 5), Rat Brain (Lane 6), Mouse kidney (Lane 7) and Rat kidney (Lane 8). The blots were probed with Anti-Sarcomeric alpha Actin/ alpha Actinin 2 Recombinant Rabbit Polyclonal Antibody (Product # 710947, 1-2 µg/mL). A 104 kDa band corresponding to Sarcomeric alpha Actin/ alpha Actinin 2 was observed only in muscle 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)
Main image
Experimental details: For immunofluorescence analysis, human skeletal muscle myoblast cells were fixed and permeabilized for detection of endogenous sarcomeric alpha actin using Anti-Sarcomeric alpha Actin/ alpha Actinin 2 Recombinant Rabbit Polyclonal Antibody (Product # 710947, 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 sarcomeric alpha actin (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 cytoskeletal localization of sarcomeric alpha actin. 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: Immunofluorescence analysis of Sarcomeric alpha actin in mouse heart tissue: Frozen sections were fixed with ice cold acetone (5 min at -20°C) and blocked with 2% BSA for 1 hour. Whole heart longitudinal sections were incubated with Anti-Sarcomeric alpha Actin/ alpha Actinin 2 Recombinant Rabbit Polyclonal Antibody (Product # 710947, 2 µg/mL) overnight at 4°C, followed by Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, Alexa Fluor® 488 conjugate (Product # A27034, 1:2000, 45 min). Nuclei (blue) were stained using SlowFade® Gold Antifade Mountant with DAPI (Product # S36938), and cytoskeletal F-actin (red) was stained using Rhodamine Phalloidin (Product # R415, 1:300). Panel a) represents staining with the matched isotype control. Panel b) shows a representative section stained for Sarcomeric alpha actin (green). The images were captured at 20X magnification.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunofluorescence analysis of Sarcomeric alpha actin in rat heart tissue: Frozen sections were fixed with ice cold acetone (5 min at -20°C) and blocked with 2% BSA for 1 hour. Whole heart longitudinal sections were incubated with Anti-Sarcomeric alpha Actin/ alpha Actinin 2 Recombinant Rabbit Polyclonal Antibody (Product # 710947, 2 µg/mL) overnight at 4°C, followed by Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, Alexa Fluor® 488 conjugate (Product # A27034, 1:2000, 45mins). Nuclei (blue) were stained using SlowFade® Gold Antifade Mountant with DAPI (Product # S36938), and cytoskeletal F-actin (red) was stained using Rhodamine Phalloidin (Product # R415, 1:300). Panel a) represents staining with the matched isotype control. Panel b) shows a representative section stained for Sarcomeric alpha actin (green). The images were captured at 20X magnification.

710947