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Synthetic peptide conjugated to KLH derived from within residues 350 - 450 of Human Occludin.
(Peptide available as ab34440.)
Our Abpromise guarantee covers the use of ab31721 in the following tested applications.
The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
|IHC-FoFr||Use at an assay dependent concentration. PubMed: 20978075|
|WB||Use a concentration of 1 µg/ml. Detects a band of approximately 63 kDa (predicted molecular weight: 59 kDa).
Abcam recommends using 3% milk as the blocking agent.
|In-Cell ELISA||Use at an assay dependent concentration. PubMed: 22689949|
|ICC/IF||Use a concentration of 1 - 2 µg/ml.|
|IHC-P||1/100. PubMed: 19828778
ab171870 - Rabbit polyclonal IgG, is suitable for use as an isotype control with this antibody.
IHC image of Occludin staining in human normal kidney 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 ab31721, 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.
*Tissue obtained from the Human Research Tissue Bank, supported by the NIHR Cambridge Biomedical Research Centre
Tissue lysates were denatured for 10-15 minutes at 90ºC. ab31721 was incubated overnight at 4ºC and the secondary antibody for 1 hour at RT. Mouse GAPDH loading control was used (red channel) at 1/5000 dilution. Blocking was performed using Licor blocking buffer.
The immunogen sequence for ab31721 shares 78% homology with the Rat Occludin protein, which is not detected in WB (lane 1). We therefore cannot guarantee reactivity with this species.
ab31721 staining Occludin in Human Breast cancer cells by ICC/IF (Immunocytochemistry/immunofluorescence). Cells were fixed with methanol/acetone (1:1) and blocked with 0.1% milk for 30 minutes at 25°C. Samples were incubated with primary antibody (1/200 in PBS + 0.1% milk) for 16 hours at 4°C. An Alexa Fluor® 594-conjugated Goat anti-rabbit polyclonal (1/1500) was used as the secondary antibody.
This blot was produced using a 4-12% Bis-tris gel under the MOPS buffer system. The gel was run at 200V for 50 minutes before being transferred onto a Nitrocellulose membrane at 30V for 70 minutes. The membrane was then blocked for an hour using 3% Milk before being incubated with ab31721 overnight at 4°C. Antibody binding was detected using an anti-rabbit antibody conjugated to HRP, and visualised using ECL development solution ab133406.