The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
Use a concentration of 5 µg/ml.
Use a concentration of 1 µg/ml. Detects a band of approximately 105 kDa (predicted molecular weight: 43 kDa).
A SLe(x)-type proteoglycan, which through high affinity, calcium-dependent interactions with E-, P- and L-selectins, mediates rapid rolling of leukocytes over vascular surfaces during the initial steps in inflammation. Critical for the initial leukocyte capture. (Microbial infection) Acts as a receptor for enterovirus 71.
Expressed on neutrophils, monocytes and most lymphocytes.
Displays complex, core-2, sialylated and fucosylated O-linked oligosaccharides, at least some of which appear to contain poly-N-acetyllactosamine with varying degrees of substitution. Mainly disialylated or neutral forms of the core-2 tetrasaccharide, Galbeta1-->4GlcNAcbeta1-->6(Galbeta1-->3)GalNAcOH. The GlcN:GalN ratio is approximately 2:1 and the Man:Fuc ratio 3:5. Contains about 14% fucose with alpha-1,3 linkage present in two forms: One species is a disialylated, monofucosylated glycan, and the other, a monosialylated, trifucosylated glycan with a polylactosamine backbone. The fucosylated forms carry the Lewis antigen and are important for interaction with selectins and for functioning in leukocyte rolling. The modification containing the sialyl Lewis X glycan is on Thr-57. No sulfated O-glycans. Some N-glycosylation. Sulfation, in conjunction with the SLe(x)-containing glycan, is necessary for P- and L-selectin binding. High affinity P-selectin binding has a preferred requirement for the isomer sulfated on both Tyr-48 and Tyr-51, whereas L-selectin binding requires predominantly sulfation on Tyr-51 with sulfation on Tyr-48 playing only a minor role. These sulfations play an important role in L- and P-selectin-mediated neutrophil recruitment, and leukocyte rolling.
CD162 was immunoprecipitated using 0.5mg Rat thymus tissue extract, 5µg of Rabbit polyclonal to CD162 and 50µl of protein G magnetic beads (+). No antibody was added to the control (-).
The antibody was incubated under agitation with Protein G beads for 10min, Rat thymus tissue extract lysate diluted in RIPA buffer was added to each sample and incubated for a further 10min under agitation.
Proteins were eluted by addition of 40µl SDS loading buffer and incubated for 10min at 70°C; 10µl of each sample was separated on a SDS PAGE gel, transferred to a nitrocellulose membrane, blocked with 5% BSA and probed with ab66882.
All lanes : Anti-CD162 antibody (ab66882) at 1 µg/ml
Lane 1 : TE 671 (Human Rhabdomyosarcoma) Whole Cell Lysate Lane 2 : HeLa (Human epithelial carcinoma cell line) Whole Cell Lysate Lane 3 : Rat Thymus Tissue Lysate
Lysates/proteins at 10 µg per lane.
Secondary Goat polyclonal to Rabbit IgG - H&L - Pre-Adsorbed (HRP) at 1/3000 dilution
Performed under reducing conditions.
Predicted band size : 43 kDa Observed band size : 105 kDa (why is the actual band size different from the predicted?) Additional bands at : 200 kDa. We are unsure as to the identity of these extra bands.CD162 contains a number of potential glycosylation and phosphorylation sites (SwissProt) which may explain its migration at a higher molecular weight than predicted.