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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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Validation data
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- Product number
- 711524 - Provider product page
- Provider
- Invitrogen Antibodies
- Product name
- PDCD4 Recombinant Polyclonal Antibody
- Antibody type
- Polyclonal
- Antigen
- Other
- Description
- This antibody is predicted to react with Monkey, Cat, Horse, Sheep
- Concentration
- 0.5 mg/mL
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Supportive validation
- Submitted by
- Invitrogen Antibodies (provider)
- Main image
- Experimental details
- Western blot analysis was performed on whole cell extracts (30 µg lysate) of HeLa (Lane 1), HEL.92.1.7 (Lane 2) and T-47D (Lane 3). The blots were probed with Anti-PDCD4 Recombinant Rabbit Polyclonal Antibody (Product # 711524, 1-2 µg/mL). A 52 kDa band corresponding to PDCD4 was observed. 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).
- Submitted by
- Invitrogen Antibodies (provider)
- Main image
- Experimental details
- Knockdown of PDCD4 was achieved by transfecting HeLa cells with PDCD4 specific siRNA (Silencer® select Cat # s26049 and s223738). Western blot analysis (Fig a) was performed using Whole cell extract from the HeLa knock down cells (lane 3), non-specific scrambled siRNA transfected cells (lane 2) and untransfected cells (lane 1). The blots were probed with Anti-PDCD4 Recombinant Rabbit Polyclonal Antibody (Product # 711524, 1-3 µg/mL) and Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, HRP conjugate (Product # A27036, 0.4 µg/mL, 1:5000 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 PDCD4.
Supportive validation
- Submitted by
- Invitrogen Antibodies (provider)
- Main image
- Experimental details
- For immunofluorescence analysis, serum starved MCF7 cells were fixed and permeabilized for detection of endogenous PDCD4 using Anti-PDCD4 Recombinant Rabbit Polyclonal Antibody (Product # 711524, 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 PDCD4 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 translocation of PDCD4 in serum starved cells. Panel e) shows cells under normal growth conditions with PDCD4 localized in the nucleus. Panel f) represents control cells with no primary antibody to assess background.
