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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
- 702479 - Provider product page
- Provider
- Invitrogen Antibodies
- Product name
- DAP5 Recombinant Rabbit Monoclonal Antibody (8H10L15)
- Antibody type
- Monoclonal
- Antigen
- Other
- Description
- This antibody is predicted to react with Bovine, Monkey, Cat
- Antibody clone number
- 8H10L15
- Concentration
- 0.5 mg/mL
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Supportive validation
- Submitted by
- Invitrogen Antibodies (provider)
- Main image
- Experimental details
- Knockdown of eIF4G2 was achieved by transfecting HEK 293 cells with eIF4G2 specific siRNA (Silencer® select Product # s4589 and Product # s4588). Western blot analysis (Fig a) was performed using whole cell lysates from the DAP5 knock down cells (lane 3), non-specific scrambled siRNA transfected cells (lane 2) and untransfected cells (lane 1). The blots were probed with Anti-DAP5 Recombinant Rabbit Monoclonal Antibody (Product # 702479, 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). Reduction of signal upon siRNA mediated knock down confirms that antibody is specific to eIF4G2
- Submitted by
- Invitrogen Antibodies (provider)
- Main image
- Experimental details
- Western blot analysis was performed on Whole cell extracts (30 µg lysate) of HEK-293 (Lane 1), SW480 (Lane 2), A549 (Lane 3) and SH-SY5Y (Lane 4). The blots were probed with Anti-DAP5 Recombinant Rabbit Monoclonal Antibody (Product # 702479, 1 µg/mL). A 102 kDa band corresponding to eIF4G2 was observed across the cell lines 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, 1:2500 dilution). Known quantity of protein samples were electrophoresed using Novex® NuPAGE® 10% Bis-Tris gel (Product # NP0301BOX), 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, HeLa cells were fixed and permeabilized for detection of endogenous DAP5 using Anti- DAP5 Recombinant Rabbit Monoclonal Antibody (Product # 702479, 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 eIF4G2 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 eIF4G2. Panel e) represents control cells with no primary antibody to assess background. The images were captured at 60X magnification.
