The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
1/1000 - 1/5000. Detects a band of approximately 87 kDa (predicted molecular weight: 85 kDa).
Use at an assay dependent concentration.
ab172730 - Rabbit monoclonal IgG, is suitable for use as an isotype control with this antibody.
Is unsuitable for ICC.
Involved in the transduction of mitogenic signals from the cell membrane to the nucleus. May play a role in the postsynaptic responses of hippocampal neuron.
Brain and testis.
Note=Defects in BRAF are found in a wide range of cancers. Defects in BRAF may be a cause of colorectal cancer (CRC) [MIM:114500]. Defects in BRAF are involved in lung cancer (LNCR) [MIM:211980]. Defects in BRAF are involved in non-Hodgkin lymphoma (NHL) [MIM:605027]. NHL is a cancer that starts in cells of the lymph system, which is part of the body's immune system. NHLs can occur at any age and are often marked by enlarged lymph nodes, fever and weight loss. Defects in BRAF are a cause of cardiofaciocutaneous syndrome (CFC syndrome) [MIM:115150]; also known as cardio-facio-cutaneous syndrome. CFC syndrome is characterized by a distinctive facial appearance, heart defects and mental retardation. Heart defects include pulmonic stenosis, atrial septal defects and hypertrophic cardiomyopathy. Some affected individuals present with ectodermal abnormalities such as sparse, friable hair, hyperkeratotic skin lesions and a generalized ichthyosis-like condition. Typical facial features are similar to Noonan syndrome. They include high forehead with bitemporal constriction, hypoplastic supraorbital ridges, downslanting palpebral fissures, a depressed nasal bridge, and posteriorly angulated ears with prominent helices. The inheritance of CFC syndrome is autosomal dominant. Defects in BRAF are the cause of Noonan syndrome type 7 (NS7) [MIM:613706]. Noonan syndrome is a disorder characterized by facial dysmorphic features such as hypertelorism, a downward eyeslant and low-set posteriorly rotated ears. Other features can include short stature, a short neck with webbing or redundancy of skin, cardiac anomalies, deafness, motor delay and variable intellectual deficits. Defects in BRAF are the cause of LEOPARD syndrome type 3 (LEOPARD3) [MIM:613707]. LEOPARD3 is a disorder characterized by lentigines, electrocardiographic conduction abnormalities, ocular hypertelorism, pulmonic stenosis, abnormalities of genitalia, retardation of growth, and sensorineural deafness. Note=A chromosomal aberration involving BRAF is found in pilocytic astrocytomas. A tandem duplication of 2 Mb at 7q34 leads to the expression of a KIAA1549-BRAF fusion protein with a constitutive kinase activity and inducing cell transformation.
Belongs to the protein kinase superfamily. TKL Ser/Thr protein kinase family. RAF subfamily. Contains 1 phorbol-ester/DAG-type zinc finger. Contains 1 protein kinase domain. Contains 1 RBD (Ras-binding) domain.
Nucleus. Cytoplasm. Cell membrane. Colocalizes with RGS14 and RAF1 in both the cytoplasm and membranes.
Western blot - Anti-B Raf antibody [EP152Y] (ab33899)
Lane 1: Wild-type HAP1 whole cell lysate (40 µg) Lane 2: B Raf knockout HAP1 whole cell lysate (40 µg) Lane 3: HeLa whole cell lysate (40 µg)
Lanes 1 - 3: Merged signal (red and green). Green - ab33899 observed at 90 kDa. Red - loading control, ab8245, observed at 37 kDa.
ab33899 was shown to recognize B Raf in wild-type HAP1 cells as signal was lost at the expected MW in B Raf knockout cells. Additional cross-reactive bands were observed in the wild-type and knockout cells. Wild-type and B Raf knockout samples were subjected to SDS-PAGE. ab33899 and ab8245 (Mouse anti-GAPDH loading control) were incubated overnight at 4°C at 1/1000 dilution and 1/20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye® 800CW) preabsorbed ab216773 and Goat anti-Mouse IgG H&L (IRDye® 680RD) preabsorbed ab216776 secondary antibodies at 1/20000 dilution for 1 hour at room temperature before imaging.
Overlay histogram showing SH-SY5Y cells stained with ab33899 (red line). The cells were fixed with 4% paraformaldehyde (10 min) and then permeabilized with 0.1% PBS-Tween for 20 min. The cells were then incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions followed by the antibody (ab33899, 1/1000 dilution) for 30 min at 22°C. The secondary antibody used was DyLight® 488 goat anti-rabbit IgG (H+L) (ab96899) at 1/500 dilution for 30 min at 22°C. Isotype control antibody (black line) was rabbit IgG (monoclonal) (0.1μg/1x106 cells) used under the same conditions. Unlabelled sample (blue line) was also used as a control. Acquisition of >5,000 events were collected using a 20mW Argon ion laser (488nm) and 525/30 bandpass filter.
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-B Raf antibody [EP152Y] (ab33899)This image is courtesy of an Abreview submitted by Sedar Balci
ab33899 staining B Raf cells from human prostate tissue by immunohistochemistry (formalin/PFA-fixed paraffin-embedded sections). Cells were formaldehyde fixed and permeabilized in PBS-Tween 20 prior to blocking in 70% serum for 10 minutes at 25°C. The primary antibody was diluted 1/250 and incubated with the sample for 1 hour at 25°C. A biotin conjugated goat polyclonal to mouse Ig was used as the secondary.
Western blot - Anti-B Raf antibody [EP152Y] (ab33899)
All lanes : Anti-B Raf antibody [EP152Y] (ab33899) at 1/1000 dilution
Lane 1 : Lysate prepared from mouse brain Lane 2 : Lysate prepared from mouse heart Lane 3 : Lysate prepared from mouse kidney Lane 4 : Lysate prepared from mouse spleen Lane 5 : Lysate prepared from rat brain Lane 6 : Lysate prepared from rat heart Lane 7 : Lysate prepared from rat kidney Lane 8 : Lysate prepared from rat spleen
Predicted band size: 85 kDa
Exposure time: 3 minutes
This product has been referenced in:
van Veen JE et al. P2A-Fluorophore Tagging of BRAF Tightly Links Expression to Fluorescence In Vivo. PLoS One11:e0157661 (2016).
Read more (PubMed: 27348307) »
Ding Z et al. Griffipavixanthone, a dimeric xanthone extracted from edible plants, inhibits tumor metastasis and proliferation via downregulation of the RAF pathway in esophageal cancer. Oncotarget7:1826-37 (2016).
Read more (PubMed: 26646323) »