The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
1/100. Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.
1/5000. Detects a band of approximately 87 kDa (predicted molecular weight: 87 kDa).
Furin is likely to represent the ubiquitous endoprotease activity within constitutive secretory pathways and capable of cleavage at the RX(K/R)R consensus motif.
Seems to be expressed ubiquitously.
Belongs to the peptidase S8 family. Furin subfamily. Contains 1 homo B/P domain.
Contains a cytoplasmic domain responsible for its TGN localization and recycling from the cell surface.
The inhibition peptide, which plays the role of an intramolecular chaperone, is autocatalytically removed in the endoplasmic reticulum (ER) and remains non-covalently bound to furin as a potent autoinhibitor. Following transport to the trans Golgi, a second cleavage within the inhibition propeptide results in propeptide dissociation and furin activation. Phosphorylation is required for TGN localization of the endoprotease. In vivo, exists as di-, mono- and non-phosphorylated forms.
Golgi apparatus > trans-Golgi network membrane. Cell membrane. Shuttles between the trans-Golgi network and the cell surface. Propeptide cleavage is a prerequisite for exit of furin molecules out of the endoplasmic reticulum (ER). A second cleavage within the propeptide occurs in the trans Golgi network (TGN), followed by the release of the propeptide and the activation of furin.
IHC image of Furin staining in a section of formalin-fixed paraffin-embedded human normal pancreas*. The section was pre-treated using pressure cooker heat mediated antigen retrieval with sodium citrate buffer (pH6) for 30mins, and incubated overnight at +4°C with ab202544 at 1/100 dilution. DAB was used as the chromogen (ab103723), diluted 1/100 and incubated for 10min at room temperature. The section was counterstained with haematoxylin and mounted with DPX. The inset negative control image is taken from an identical assay without primary antibody.
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
Western blot - Anti-Furin antibody [EPR14674] (HRP) (ab202544)
All lanes : Anti-Furin antibody [EPR14674] (HRP) (ab202544) at 1/5000 dilution
Lane 1 : HepG2 (Human hepatocellular liver carcinoma cell line) Whole Cell Lysate Lane 2 : HeLa (Human epithelial carcinoma cell line) Whole Cell Lysate Lane 3 : U-87 MG (Human glioblastoma astrocytoma) Whole Cell Lysate Lane 4 : Caco 2 (Human colonic carcinoma cell line) Whole Cell Lysate
Lysates/proteins at 10 µg per lane.
Developed using the ECL technique.
Performed under reducing conditions.
Predicted band size: 87 kDa Observed band size: 87 kDa
Exposure time: 20 minutes
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 ab202544 overnight at 4°C. Antibody binding was visualised using ECL development solution ab133406.