Antibody data
- Antibody Data
- Antigen structure
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- Validations
- Western blot [2]
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Validation data
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- Product number
- PA1-21440 - Provider product page
- Provider
- Invitrogen Antibodies
- Product name
- A-Raf Polyclonal Antibody
- Antibody type
- Polyclonal
- Antigen
- Synthetic peptide
- Description
- Recommended positive controls: MAD109 cells.
- Reactivity
- Human, Mouse, Rat
- Host
- Rabbit
- Isotype
- IgG
- Vial size
- 250 µg
- Concentration
- 1.0 mg/mL
- Storage
- Store at 4°C short term. For long term storage, store at -20°C, avoiding freeze/thaw cycles.
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Supportive validation
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- Invitrogen Antibodies (provider)
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- Experimental details
- Western blot analysis was performed on membrane enriched extracts (30 µg lysate) of HeLa (1), A-431 (2), C2C12 (3), SK-N-SH (4), HT-29 (5) and NIH/3T3 (6). The blot was probed with Rabbit Anti-A-Raf Polyclonal Antibody (Product # PA1-21440, 1 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). 68 kDa band corresponding to A-Raf was observed across the cell lines tested. 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® 2 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 A-Raf was achieved by transfecting HeLa cells with A-Raf specific validated siRNAs (Silencer® select Product # s577). Western blot analysis (Fig a) was performed using membrane enriched extracts from the A-Raf knockdown cells (lane 3), non-specific scrambled siRNA transfected cells (lane 2) and untransfected cells (lane 1). The blots were probed with Rabbit Anti-A-Raf Polyclonal Antibody (Product # PA1-21440, 1 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 A-Raf.