This antibody gave a positive signal in the following Lysates:
HeLa Whole Cell - Hydroxyurea Treated (48hr, 2uM), HeLa Whole Cell - Bleomycin Treated (20U/ml), HeLa Whole Cell - Staurosporine Treated (48hr, 500nM), Y79 Whole Cell, Mouse Skeletal Muscle Tissue, HeLa Whole Cell, SK N SH Whole Cell
This antibody gave a positive result when used in the following methanol fixed cell lines: SV40LT-SMC
IHC-P: FFPE human heart tissue sections.
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 1 µg/ml. Detects a band of approximately 60 kDa (predicted molecular weight: 42 kDa).
Use a concentration of 1 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 5 µg/ml.
Plays pivotal roles in spermatogenesis, embryo implantation, neural network formation and tumor progression. Stimulates adjacent fibroblasts to produce matrix metalloproteinases (MMPS). May target monocarboxylate transporters SLC16A1, SLC16A3 and SLC16A8 to plasma membranes of retinal pigment epithelium and neural retina. Seems to be a receptor for oligomannosidic glycans. In vitro, promotes outgrowth of astrocytic processes.
Present only in vascular endothelium in non-neoplastic regions of the brain, whereas it is present in tumor cells but not in proliferating blood vessels in malignant gliomas.
IHC image of CD147 staining in human heart 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 ab64616,1µg/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.
ab64616 stained SV40LT-SMC cells. The cells were 100% methanol fixed (5 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 ab64616 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.
CD147 was immunoprecipitated using 0.5mg Hela whole cell extract, 5µg of Rabbit polyclonal to CD147 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, Hela whole cell 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 ab64616.
All lanes : Anti-CD147 antibody (ab64616) at 1 µg/ml
Lane 1 : HeLa Whole Cell Lysate - Hydroxyurea Treated (48hr, 2uM) Lane 2 : HeLa Whole Cell Lysate - Bleomycin Treated (20U/ml) Lane 3 : HeLa Whole Cell Lysate - Staurosporine Treated (48hr, 500nM) Lane 4 : Y79 (Human retinoblastoma cell line) Whole Cell Lysate Lane 5 : Skeletal Muscle (Mouse) Tissue Lysate Lane 6 : HeLa (Human epithelial carcinoma cell line) Whole Cell Lysate Lane 7 : SK N SH (Human neuroblastoma) Whole Cell 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 : 42 kDa Observed band size : 60 kDa (why is the actual band size different from the predicted?) Additional bands at : 150 kDa. We are unsure as to the identity of these extra bands.CD147 contains known glycosylation sites (SwissProt) which may explain its migration at a higher molecular weight than predicted.