The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
Use a concentration of 5 µg/ml.
1/100 - 1/500. Detects a band of approximately 58 kDa (predicted molecular weight: 50 kDa).
Tyrosine kinase substrate that inhibits growth-factor-mediated activation of MAP kinase. Negatively regulates hematopoiesis of bone marrow.
Weakly expressed in embryonic cell line (HEK-293).
Defects in SPRED1 are the cause of Legius syndrome (LEGIUSS) [MIM:611431]. It is a disorder characterized mainly by cafe au lait macules without neurofibromas or other tumor manifestations of neurofibromatosis type 1, axillary freckling, and macrocephaly. Additional clinical manifestations include Noonan-like facial dysmorphism, lipomas, learning disabilities and attention deficit-hyperactivity.
ICC/IF image of ab62911 stained A549 cells. The cells were 4% formaldehyde 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 ab62911 at 5µg/ml overnight at +4°C. The secondary antibody (green) was DyLight® 488 goat anti- rabbit (ab96899) IgG (H+L) used at a 1/250 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.
Western blot - SPRED1 antibody (ab62911)
All lanes : Anti-SPRED1 antibody (ab62911) at 1/100 dilution
Lane 1 : 293 cell lysates nontransfected Lane 2 : 293 cell lysates transiently transfected with the SPRED1 gene
Lysates/proteins at 2 µg per lane.
Predicted band size : 50 kDa Observed band size : 58 kDa (why is the actual band size different from the predicted?) Additional bands at : 40 kDa. We are unsure as to the identity of these extra bands.There are bands of 53 kDa and 40 kDa in the nontransfected 293 cell lysates. We are unsure as to the identity of these bands.