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 100 kDa (predicted molecular weight: 75 kDa).
Use a concentration of 1 µg/ml.
Use a concentration of 1 µg/ml.
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.
Predicted band size : 75 kDa Observed band size : 100 kDa (why is the actual band size different from the predicted?) Although the predicted band size is 75kDa based on Swiss-prot data, a band of 120kDa has been previously observed. Mol Cell Biol. 2002 Dec;22(23):8267-77 PMID: 12417729. The band seen at 100kDa is also consistent with the banding pattern observed for other commercially available antibodies to Rad21.
Western blot - Anti-Rad21 antibody (ab42522)
All lanes : Anti-Rad21 antibody (ab42522) at 1 µg/ml
Lane 1 : Brain (Mouse) Tissue Lysate Lane 2 : Kidney (Mouse) Tissue Lysate Lane 3 : Heart (Mouse) Tissue Lysate Lane 4 : Thymus (Mouse) Tissue Lysate
Lysates/proteins at 10 µg per lane.
Secondary Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/10000 dilution Developed using the ECL technique
The predicted molecular weight of Rad21 is 72 kDa (SwissProt), however we expect to observe a banding pattern at 100 kDa. Abcam welcomes customer feedback and would appreciate any comments regarding this product and the data presented above.
ICC/IF image of ab42522 stained human HeLa cells. The cells were PFA fixed (10 min), permabilised in TBS-T (20 min) and incubated with the antibody (ab42522, 1µg/ml) for 1h at room temperature. 1%BSA / 10% normal goat serum / 0.3M glycine was used to quench autofluorescence and block non-specific protein-protein interactions. The secondary antibody (green) was Alexa Fluor® 488 goat anti-rabbit IgG (H+L) used at a 1/1000 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (red). DAPI was used to stain the cell nuclei (blue).
IHC image of ab42522 staining Rad21 in Human normal breast formalin fixed paraffin embedded tissue section, performed on a Leica BondTM 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 ab42522, 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.