Anti-Raf1 antibody [EP4969] ab181115 is a rabbit monoclonal antibody that is used in Raf1 western blotting, immunofluorescence and flow cytometry. Suitable for human, mouse and rat samples.
- Recombinant format for unrivaled batch-batch consistency: no need for same-lot requests
- Specificity confirmed with RAF1 knockout cell line validation
IgG
Rabbit
Preservative: 0.01% Sodium azide
Constituents: PBS, 40% Glycerol (glycerin, glycerine), 0.05% BSA
Liquid
Monoclonal
WB | ICC/IF | Flow Cyt (Intra) | |
---|---|---|---|
Human | Tested | Tested | Tested |
Mouse | Tested | Expected | Expected |
Rat | Tested | Expected | Expected |
Species | Dilution info | Notes |
---|---|---|
Species Mouse | Dilution info 1/1000 | Notes - |
Species Rat | Dilution info 1/1000 | Notes - |
Species Human | Dilution info 1/1000 | Notes - |
Species | Dilution info | Notes |
---|---|---|
Species Human | Dilution info 1/100 - 1/250 | Notes - |
Species | Dilution info | Notes |
---|---|---|
Species Mouse, Rat | Dilution info Use at an assay dependent concentration. | Notes - |
Species | Dilution info | Notes |
---|---|---|
Species Human | Dilution info 1/100 | Notes - |
Species | Dilution info | Notes |
---|---|---|
Species Mouse, Rat | Dilution info Use at an assay dependent concentration. | Notes - |
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Serine/threonine-protein kinase that acts as a regulatory link between the membrane-associated Ras GTPases and the MAPK/ERK cascade, and this critical regulatory link functions as a switch determining cell fate decisions including proliferation, differentiation, apoptosis, survival and oncogenic transformation. RAF1 activation initiates a mitogen-activated protein kinase (MAPK) cascade that comprises a sequential phosphorylation of the dual-specific MAPK kinases (MAP2K1/MEK1 and MAP2K2/MEK2) and the extracellular signal-regulated kinases (MAPK3/ERK1 and MAPK1/ERK2). The phosphorylated form of RAF1 (on residues Ser-338 and Ser-339, by PAK1) phosphorylates BAD/Bcl2-antagonist of cell death at 'Ser-75'. Phosphorylates adenylyl cyclases: ADCY2, ADCY5 and ADCY6, resulting in their activation. Phosphorylates PPP1R12A resulting in inhibition of the phosphatase activity. Phosphorylates TNNT2/cardiac muscle troponin T. Can promote NF-kB activation and inhibit signal transducers involved in motility (ROCK2), apoptosis (MAP3K5/ASK1 and STK3/MST2), proliferation and angiogenesis (RB1). Can protect cells from apoptosis also by translocating to the mitochondria where it binds BCL2 and displaces BAD/Bcl2-antagonist of cell death. Regulates Rho signaling and migration, and is required for normal wound healing. Plays a role in the oncogenic transformation of epithelial cells via repression of the TJ protein, occludin (OCLN) by inducing the up-regulation of a transcriptional repressor SNAI2/SLUG, which induces down-regulation of OCLN. Restricts caspase activation in response to selected stimuli, notably Fas stimulation, pathogen-mediated macrophage apoptosis, and erythroid differentiation.
RAF, RAF1, RAF, RAF proto-oncogene serine/threonine-protein kinase, Proto-oncogene c-RAF, Raf-1, cRaf
Anti-Raf1 antibody [EP4969] ab181115 is a rabbit monoclonal antibody that is used in Raf1 western blotting, immunofluorescence and flow cytometry. Suitable for human, mouse and rat samples.
- Recombinant format for unrivaled batch-batch consistency: no need for same-lot requests
- Specificity confirmed with RAF1 knockout cell line validation
IgG
Rabbit
Preservative: 0.01% Sodium azide
Constituents: PBS, 40% Glycerol (glycerin, glycerine), 0.05% BSA
Liquid
Monoclonal
EP4969
Affinity purification Protein A
Blue Ice
1-2 weeks
+4°C
-20°C
Upon delivery aliquot
Avoid freeze / thaw cycle
Our RabMAb® technology is a patented hybridoma-based technology for making rabbit monoclonal antibodies. For details on our patents, please refer to RabMAb® patents.
This product is a recombinant monoclonal antibody, which offers several advantages including:
For more information, read more on recombinant antibodies.
This supplementary information is collated from multiple sources and compiled automatically.
Raf1 also known as c-Raf or Raf-1 is a serine/threonine-protein kinase that plays an important role in cell division differentiation and survival. Its molecular weight is approximately 74 kDa. Raf1 is part of the MAPK/ERK signaling pathway and is ubiquitously expressed in many tissues including the brain heart and liver. As a component of the signaling cascade its activation translates extracellular signals into intracellular responses.
Raf1 regulates important cellular processes by activating downstream kinases in response to external stimuli. Raf1 forms a complex with other proteins such as Ras facilitating its role as an essential component of the MAPK/ERK pathway. Upon activation by Ras Raf1 phosphorylates and activates MEK1 and MEK2 which in turn activate the ERK1 and ERK2. This signaling axis is involved in controlling gene expression and cellular proliferation.
Raf1 is integrally involved in the MAPK/ERK pathway which is critical for transducing signals from growth factors and mitogens. It relates closely with proteins such as Ras MEK and ERK in this pathway. The pathway is important for regulating cellular responses to various stimuli and is particularly involved in processes such as cell cycle control and apoptosis.
Raf1 has associations with certain types of cancer and Noonan syndrome. In various cancers mutations or dysregulation of Raf1 or related components such as Ras can lead to uncontrolled cellular proliferation. In Noonan syndrome Raf1 mutations result in anomalies in the Ras/MAPK pathway which can impact normal development. Raf1’s interactions in these pathways indicate its relevance as a potential target for therapeutic intervention.
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We have not tested this specific species and application combination in-house, but expect it will work. It is covered by our product promise.
This species and application combination has not been tested, but we predict it will work based on strong homology. However, this combination is not covered by our product promise.
We do not recommend this combination. It is not covered by our product promise.
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Lanes 1- 2: Merged signal (red and green). Green - ab181115 observed at 73 kDa. Red - Anti-GAPDH antibody [6C5] - Loading Control (Anti-GAPDH antibody [6C5] - Loading Control ab8245) observed at 37 kDa.
ab181115 was shown to react with Raf1 in wild-type HeLa cells in western blot. Loss of signal was observed when knockout cell line Human RAF1 knockout HeLa cell line ab264978 (knockout cell lysate Human RAF1 knockout HeLa cell lysate ab257126) was used. Wild-type HeLa and Raf1 knockout HeLa cell lysates were subjected to SDS-PAGE. Membrane was blocked for 1 hour at room temperature in 0.1% TBST with 3% non-fat dried milk. ab181115 and Anti-GAPDH antibody [6C5] - Loading Control (Anti-GAPDH antibody [6C5] - Loading Control ab8245) overnight at 4°C at a 1 in 20000 dilution and a 1 in 20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye®800CW) preadsorbed (Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed ab216773) and Goat anti-Mouse IgG H&L (IRDye®680RD) preadsorbed (Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed ab216776) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.
All lanes: Western blot - Anti-Raf1 antibody [EP4969] - N-terminal (ab181115) at 1/20000 dilution
Lane 1: Wild-type HeLa cell lysate at 20 µg
Lane 2: RAF1 knockout HeLa cell lysate at 20 µg
Performed under reducing conditions.
Predicted band size: 73 kDa
Observed band size: 73 kDa
Immunocytochemistry/ Immunofluorescence analysis of HeLa (Human cervix adenocarcinoma epithelial cell) cells labeling Raf1 with purified ab181115 at 1/100 dilution (10 μg/ml). Cells were fixed in 4% Paraformaldehyde and permeabilized with 0.1% tritonX-100. Cells were counterstained with Alexa Fluor® 594 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker ab195889 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor® 594) 1/200 (2.5 μg/ml). Goat anti rabbit IgG (Alexa Fluor® 488, Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) ab150077) was used as the secondary antibody at 1/1000 (2 μg/ml) dilution. DAPI (blue) was used as nuclear counterstain. PBS instead of the primary antibody was used as the secondary antibody only control.
Lanes 1: Wild-type HAP1 cell lysate (20 μg)
Lanes 2: Raf1 knockout HAP1 cell lysate (20 μg)
Lanes 1 and 2: Merged signal (red and green).
Green - Target observed at 74 kDa. Red - loading control, Anti-beta Actin antibody [mAbcam 8226] - Loading Control ab8226, observed at 42 kDa
This western blot image is a comparison between unpurified ab181115 and a competitor's top cited discontinued rabbit polyclonal antibody.
All lanes: Western blot - Anti-Raf1 antibody [EP4969] - N-terminal (ab181115)
Predicted band size: 73 kDa
Intracellular Flow Cytometry analysis of K-562 (Human chronic myelogenous leukemia lymphoblast) cells labelling with unpurified ab181115 at 1/100 dilution (10.79μg/mL) (red). Cells were fixed with 4% paraformaldehyde . Goat anti rabbit IgG (Alexa Fluor® 488, Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) ab150077) was used as the secondary antibody at 1/2000 dilution. Isotype control - 90% methanol . Unlabeled control - Rabbit monoclonal IgG (Rabbit IgG, monoclonal [EPR25A] - Isotype Control ab172730) / Black.
All lanes: Western blot - Anti-Raf1 antibody [EP4969] - N-terminal (ab181115) at 1/1000 dilution
All lanes: PC-12 (Rat adrenal gland pheochromocytoma) whole cell lysates at 20 µg
All lanes: Western blot - Goat Anti-Rabbit IgG H&L (HRP) (Goat Anti-Rabbit IgG H&L (HRP) ab97051) at 1/20000 dilution
Predicted band size: 73 kDa
Observed band size: 73 kDa
Lanes 1 and 2: Green signal from target – ab181115 observed at 74 kDa
Lanes 3 and 4: Red signal from loading control – Anti-beta Actin antibody [mAbcam 8226] - Loading Control ab8226 observed at 42 kDa
Lanes 5 and 6: Merged (red and green) signal
Unpurified ab181115 was shown to specifically react with Raf1 when Raf1 knockout samples were used. Wild-type and Raf1 knockout samples were subjected to SDS-PAGE. ab181115 and Anti-beta Actin antibody [mAbcam 8226] - Loading Control ab8226 (loading control to beta actin) were both diluted 1/1000 and incubated overnight at 4°C. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed ab216773) and Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed (Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed ab216776) secondary antibodies at 1/10000 dilution for 1 hour at room temperature before imaging.
Lanes 1 - 4: Western blot - Anti-Raf1 antibody [EP4969] - N-terminal (ab181115) at 1/1000 dilution
Lanes 5 - 6: Western blot - Anti-Raf1 antibody [EP4969] - N-terminal (ab181115)
Lanes 1, 3 and 5: Wild-type HAP1 cell lysate at 20 µg
Lanes 2, 4 and 6: Raf1 knockout HAP1 cell lysate at 20 µg
Predicted band size: 73 kDa
All lanes: Western blot - Anti-Raf1 antibody [EP4969] - N-terminal (ab181115) at 1/1000 dilution
Lane 1: RAW 264.7(Mouse Abelson murine leukemia virus-induced tumor macrophage) whole cell lysates at 20 µg
Lane 2: C2C12 (Mouse myoblasts myoblast) whole cell lysates at 20 µg
Lane 3: NIH/3T3 (Mouse embryonic fibroblast) whole cell lysates at 20 µg
All lanes: Western blot - Goat Anti-Rabbit IgG H&L (HRP) (Goat Anti-Rabbit IgG H&L (HRP) ab97051) at 1/20000 dilution
Predicted band size: 73 kDa
Observed band size: 73 kDa
All lanes: Western blot - Anti-Raf1 antibody [EP4969] - N-terminal (ab181115) at 1/1000 dilution
Lane 1: HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysates at 20 µg
Lane 2: K-562 (Human chronic myelogenous leukemia lymphoblast) whole cell lysates at 20 µg
All lanes: Western blot - Goat Anti-Rabbit IgG H&L (HRP) (Goat Anti-Rabbit IgG H&L (HRP) ab97051) at 1/20000 dilution
Predicted band size: 73 kDa
Observed band size: 73 kDa
All lanes: Western blot - Anti-Raf1 antibody [EP4969] - N-terminal (ab181115) at 1/1000 dilution
Lane 1: HeLa cell lysate at 20 µg
Lane 2: K562 cell lysate at 20 µg
All lanes: Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugate at 1/1000 dilution
Predicted band size: 73 kDa
Observed band size: 73 kDa
Immunofluorescent analysis of acetone-fixed K562 cells labeling Raf1 with unpurified ab181115 at 1/250 dilution, followed by Goat anti rabbit IgG (Alexa Fluor® 488) secondary antibody at 1/200 dilution. Counter stained with Dapi.
Western blot: Anti-RAF1 antibody [EP4969] (ab181115) staining at 1/1000 dilution, shown in green; Mouse anti-GAPDH antibody [6C5] (Anti-GAPDH antibody [6C5] - Loading Control ab8245) loading control staining at 1/20000 dilution, shown in red. In Western blot, ab181115 was shown to bind specifically to RAF1. A band was observed at 73 kDa in wild-type HCT 116 cell lysates with no signal observed at this size in RAF1 knockout cell line. To generate this image, wild-type and RAF1 knockout HCT 116 cell lysates were analysed. First, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 3 % milk in TBS-0.1 % Tween® 20 (TBS-T) before incubation with primary antibodies overnight at 4 °C. Blots were washed four times in TBS-T, incubated with secondary antibodies for 1 h at room temperature, washed again four times then imaged. Secondary antibodies used were Goat anti-Rabbit IgG H&L 800CW and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution.
All lanes: Western blot - Anti-Raf1 antibody [EP4969] - N-terminal (ab181115) at 1/1000 dilution
Lane 1: Wild-type HCT 116 cell lysate at 20 µg
Lane 2: RAF1 knockout HCT 116 cell lysate at 20 µg
All lanes: Goat anti-Rabbit IgG H&L 800CW and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution
Performed under reducing conditions.
Predicted band size: 73 kDa
Observed band size: 73 kDa
Anti-GAPDH antibody [EPR16891] - Loading Control ab181602 was used as GAPDH loading control.
ab181115 was used to detect Raf1.
Anti-Raf1 (phospho S43) antibody [EPR5692(2)] ab150365 was used to detect Raf1 phospho S43.
All lanes: Purified at 1/5000 dilution
Lane 1: HEK-293 (Human embryonic kidney epithelial cell) whole cell lysates at 15 µg
Lane 2: HEK-293 (Human embryonic kidney epithelial cell) treated with 100nM Phorbol-12-myristate-13-acetate for 1 hour whole cell lysates at 15 µg
Lane 3: HEK-293 (Human embryonic kidney epithelial cell) treated with 100nM Phorbol-12-myristate-13-acetate for 1 hour whole cell lysates, then incubated with phosphatase at 15 µg
All lanes: Western blot - Goat Anti-Rabbit IgG H&L (HRP) (Goat Anti-Rabbit IgG H&L (HRP) ab97051) at 1/20000 dilution
Predicted band size: 73 kDa
Observed band size: 73 kDa
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