This antibody gave a positive signal in the following tissue lysates:
Human Brain; Human Testis; Human Liver.
Whole cell lysates: MBA MD 231; MDA MB 361; MCF7; T47D.
This antibody gave a positive result in IHC in the following FFPE tissue: Human Testis.
Shipped at 4°C. Store at +4°C short term (1-2 weeks). Upon delivery aliquot. Store at -20°C or -80°C. Avoid freeze / thaw cycle.
The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
Use a concentration of 0.1 µg/ml. Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.
Use a concentration of 1 µg/ml. Detects a band of approximately 100 kDa (predicted molecular weight: 93 kDa).
Use a concentration of 1 µg/ml.
Docking protein which plays a central coordinating role for tyrosine kinase-based signaling related to cell adhesion. Implicated in induction of cell migration. Overexpression confers antiestrogen resistance on breast cancer cells.
Widely expressed with an abundant expression in the testis. Low level of expression seen in the liver, thymus, and peripheral blood leukocytes. The protein has been detected in a B-cell line.
Belongs to the CAS family. Contains 1 SH3 domain.
Contains a central domain (substrate domain) containing multiple potential SH2-binding sites and a C-terminal domain containing a divergent helix-loop-helix (HLH) motif. The SH2-binding sites putatively bind CRK, NCK and ABL1 SH2 domains. The HLH motif is absolutely required for the induction of pseudohyphal growth in yeast and mediates heterodimerization with NEDD9. A serine-rich region promotes activation of the serum response element (SRE). The SH3 domain is necessary for the localization of the protein to focal adhesions and interacts with one proline-rich region of PTK2/FAK11.
PTK2/FAK1 activation mediates phosphorylation at the YDYVHL motif; phosphorylation is most likely catalyzed by SRC family members. SRC-family kinases are recruited to the phosphorylated sites and can phosphorylate other tyrosine residues. Tyrosine phosphorylation is triggered by integrin-mediated adhesion of cells to the extracellular matrix. Dephosphorylated by PTPN14 at Tyr-128.
Cell junction, focal adhesion. Cytoplasm. Unphosphorylated form localizes in the cytoplasm and can move to the membrane upon tyrosine phosphorylation.
IHC image of BCAR1 staining in Human Testis formalin fixed paraffin embedded tissue section, performed on a Leica Bond™ system using the standard protocol F. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20 mins. The section was then incubated with ab80016, 0.1 µg/ml, for 15 mins at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX.
For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.
Western blot - BCAR1 antibody (ab80016)
All lanes : Anti-BCAR1 antibody (ab80016) at 1 µg/ml
Lane 1 : Human breast tissue lysate - total protein (ab30090) Lane 2 : Human testis tissue lysate - total protein (ab30257) Lane 3 : Human liver tissue lysate - total protein (ab29889) Lane 4 : MDA-MB-231 (Human breast adenocarcinoma cell line) Whole Cell Lysate Lane 5 : MDA-MB-361 (Human breast adenocarcinoma cell line) Whole Cell Lysate Lane 6 : MCF7 (Human breast adenocarcinoma cell line) Whole Cell Lysate Lane 7 : T47D whole cell lysate (ab14899)
Lysates/proteins at 10 µg per lane.
Secondary Goat polyclonal to Rabbit IgG - H&L - Pre-Adsorbed (HRP) at 1/3000 dilution Developed using the ECL technique
Exposure time : 30 seconds Breast cancer anti-estrogen resistance protein 1
(BCAR1) contains a number of potential phosphorylation sites (SwissProt) which may explain its migration at a higher (100 kDa) molecular weight than predicted (93 kDa).
ICC/IF image of ab80016 stained HeLa cells. The cells were 4% PFA fixed (10 min) and then incubated in 1%BSA / 10% normal Goat serum / 0.3M glycine in 0.1% PBS-Tween for 1h to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab80016, 1µg/ml) overnight at +4°C. The secondary antibody (green) was Alexa Fluor® 488 Goat anti-Rabbit IgG (H+L) used at a 1/1000 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (red) at a 1/200 dilution for 1h. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43µM. This antibody also gave a positive result in 4% PFA fixed (10 min) HEK293, HepG2, and MCF-7 cells at 1µg/ml, and in 100% Methanol fixed (5 min) HeLa, HEK293, HepG2, and MCF-7 cells at 1µg/ml.