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Synthetic peptide corresponding to an internal sequence of human eNOS.
Our Abpromise guarantee covers the use of ab66127 in the following tested applications.
The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
|WB||1/200. Detects a band of approximately 140 kDa (predicted molecular weight: 133 kDa).
In some samples containing lower levels of endogenous eNOS, IP needs to be performed to enrich samples.
Because eNOS is of higher MW (140 kDa), it is possible that the Immunoprecipitated protein does not transfer sufficiently from gel to membrane. We suggest to transfer the protein overnight at 4oC.
Blocking is recommended with 3% BSA
|IP||Use a concentration of 2 - 5 µg/ml.|
|ELISA||Use a concentration of 0.01 - 0.1 µg/ml.|
|IHC-P||1/50 - 1/200. Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol. The recommended dilutions were determined for a 10-minute incubation at room temperature. Dilute further if results show high background.|
|ICC/IF||Use a concentration of 5 µg/ml.|
|IHC-Fr||Use at an assay dependent concentration.|
IHC image of eNOS staining in human placenta*, 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 ab66127, 1/100 dilution, 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.
*Tissue obtained from the Human Research Tissue Bank, supported by the NIHR Cambridge Biomedical Research Centre
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