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Western blotAntibody data
- Antibody Data
- Antigen structure
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- Validations
- Western blot [2]
- Immunocytochemistry [1]
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- Product number
- 702454 - Provider product page
- Provider
- Invitrogen Antibodies
- Product name
- ALPL Recombinant Rabbit Monoclonal Antibody (7H11L3)
- Antibody type
- Monoclonal
- Antigen
- Other
- Description
- This antibody is predicted to react with Monkey, Cat, Bovine, Rat
- Antibody clone number
- 7H11L3
- Concentration
- 0.5 mg/mL
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Supportive validation
- Submitted by
- Invitrogen Antibodies (provider)
- Main image
- Experimental details
- Knockdown of TNAP was achieved by transfecting U-2 OS cells with TNAP specific siRNA (Silencer® select Product # n349283 & Product # n349276). Western blot analysis (Fig a) was performed using membrane enriched extracts from TNAP knockdown cells (Lane 3), non-specific scrambled siRNA transfected cells (Lane 2) and untransfected cells (Lane 1). The blots were probed with Anti-TNAP Recombinant Rabbit Monoclonal Antibody (Product # 702454, 1-3 µg/mL) and Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, HRP conjugate (Product # A27036, 0.25 µg/mL, 1:4000 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 TNAP.
- Submitted by
- Invitrogen Antibodies (provider)
- Main image
- Experimental details
- Western blot analysis was performed on Membrane enriched extracts (30 µg lysate) of Caco-2 (Lane 4), HeLa (Lane 5) U-2 OS (Lane 6) and tissue extracts of Rat Kidney (Lane 1), Mouse Kidney (Lane 2) and Rat Liver (Lane 3). The blots were probed with Anti-TNAP Recombinant Rabbit Monoclonal Antibody (Product # 702454, 2.5 µg/mL) and detected by chemiluminescence using Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, HRP conjugate (Product # A27036, 0.25 µg/mL, 1:4000 dilution). 70 and 80 kDa band corresponding to TNAP was observed respectively across the cell lines and tissues 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, SH-SY5Y cells were fixed for detection of endogenous TNAP using Anti- TNAP Recombinant Rabbit Monoclonal Antibody (Product # 702454, 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 TNAP 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 membrane localization of TNAP. Panel e) represents control cells with no primary antibody to assess background. The images were captured at 60X magnification.
