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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
- 702747 - Provider product page
- Provider
- Invitrogen Antibodies
- Product name
- LDHA Recombinant Rabbit Monoclonal Antibody (4H18L6)
- Antibody type
- Monoclonal
- Antigen
- Synthetic peptide
- Description
- This antibody is predicted to react with Monkey, Cat, Pig
- Antibody clone number
- 4H18L6
- 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 Membrane enriched extracts (30 µg lysate) of PC-3 (Lane 1), DU 145 (Lane 2), LNCaP (Lane 3), MDA-MB-231 (Lane 4), PANC-1 (Lane 5), SK-OV-3 (Lane 6) and HeLa (Lane 7). The blots were probed with Anti-LDHA Recombinant Rabbit Monoclonal Antibody (Product # 702747, 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). A 35 kDa band corresponding to LDHA was observed across the cell lines 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).
- Submitted by
- Invitrogen Antibodies (provider)
- Main image
- Experimental details
- Knockdown of LDHA was achieved by transfecting PC-3 with LDHA specific siRNAs (Silencer® select Product # S350, S351). Western blot analysis (Fig. a) was performed using Whole Cell Extract-WCL from the LDHA knockdown cells (lane 3), non-targeting scrambled siRNA transfected cells (lane 2) and untransfected cells (lane 1). The blot was probed with LDHA Recombinant Rabbit Monoclonal Antibody (4H18L6) (Product # 702747, 1 ug/ml ) and Goat anti-Rabbit IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A27036, 1:4000 dilution). Densitometric analysis of this western blot is shown in histogram (Fig. b). Decrease in signal upon siRNA mediated knock down confirms that antibody is specific to LDHA.
Supportive validation
- Submitted by
- Invitrogen Antibodies (provider)
- Main image
- Experimental details
- For immunofluorescence analysis, PC3 cells were fixed and permeabilized for detection of endogenous LDHA using Anti- LDHA Recombinant Rabbit Monoclonal Antibody (Product # 702747, 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 LDHA 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 LDHA. Panel e) represents control cells with no primary antibody to assess background. The images were captured at 60X magnification.
