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 1 µg/ml. Detects a band of approximately 72 kDa (predicted molecular weight: 72 kDa).
Cleavable component of the cohesin complex, involved in chromosome cohesion during cell cycle, in DNA repair, and in apoptosis. The cohesin complex is required for the cohesion of sister chromatids after DNA replication. The cohesin complex apparently forms a large proteinaceous ring within which sister chromatids can be trapped. At metaphase-anaphase transition, this protein is cleaved by separase/ESPL1 and dissociates from chromatin, allowing sister chromatids to segregate. The cohesin complex may also play a role in spindle pole assembly during mitosis. Also plays a role in apoptosis, via its cleavage by caspase-3/CASP3 or caspase-7/CASP7 during early steps of apoptosis: the C-terminal 64 kDa cleavage product may act as a nuclear signal to initiate cytoplasmic events involved in the apoptotic pathway.
Belongs to the rad21 family.
The C-terminal part associates with the head of SMC1A, while the N-terminal part binds to the head of SMC3.
Cleaved by separase/ESPL1 at the onset of anaphase. Cleaved by caspase-3 and caspase-7 at the beginning of apoptosis. The cleavage by ESPL1 and caspase-3 take place at different sites. Phosphorylated; becomes hyperphosphorylated in M phase of cell cycle. The large dissociation of cohesin from chromosome arms during prophase may be partly due to its phosphorylation by PLK.
Nucleus. Chromosome. Chromosome > centromere. Associates with chromatin. Before prophase it is scattered along chromosome arms. During prophase, most of cohesin complexes dissociate from chromatin probably because of phosphorylation by PLK, except at centromeres, where cohesin complexes remain. At anaphase, it is cleaved by separase/ESPL1, leading to the dissociation of the complex from chromosomes, allowing chromosome separation. Once cleaved by caspase-3, the C-terminal 64 kDa cleavage product translocates to the cytoplasm, where it may trigger apoptosis.
IHC image of Rad21 staining in human breast fibroadenoma 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 ab153765, 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.