The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
Use a concentration of 5 µg/ml. Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.
Use a concentration of 5 µg/ml.
Use a concentration of 1 µg/ml. Detects a band of approximately 42 kDa (predicted molecular weight: 42 kDa).
The soluble form is chemotactic for T-cells and monocytes, but not for neutrophils. The membrane-bound form promotes adhesion of those leukocytes to endothelial cells. May play a role in regulating leukocyte adhesion and migration processes at the endothelium. Binds to CX3CR1.
Small intestine, colon, testis, prostate, heart, brain, lung, skeletal muscle, kidney and pancreas.
Belongs to the intercrine delta family.
A soluble short 95 kDa form may be released by proteolytic cleavage from the long membrane-anchored form. O-glycosylated with core 1 or possibly core 8 glycans.
IHC image of CX3CL1 staining in human lung 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 ab85034, 5µg/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 - CX3CL1 antibody (ab85034)
All lanes : Anti-CX3CL1 antibody (ab85034) at 1 µg/ml
Lane 1 : Human brain tissue lysate - total protein (ab29466) Lane 2 : Human heart tissue lysate - total protein (ab29431) Lane 3 : Human skeletal muscle tissue lysate - total protein (ab29330)
Lysates/proteins at 10 µg per lane.
Secondary All lanes : Goat polyclonal to Rabbit IgG - H&L - Pre-Adsorbed (HRP) at 1/3000 dilution
Developed using the ECL technique.
Performed under reducing conditions.
Predicted band size: 42 kDa Observed band size: 42 kDa Additional bands at: 50 kDa, 53 kDa. We are unsure as to the identity of these extra bands.
Exposure time: 3 minutes
CX3CL1 has 3 potential glycosylation sites (Swissprot). This might explain the additional bands observed at approximately 50-kDa and 53-kDa.
ICC/IF image of ab85034 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 (ab85034, 5µg/ml) overnight at +4°C. The secondary antibody (green) was ab96899 Dylight 488 goat anti-rabbit 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.