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 76 kDa (predicted molecular weight: 73 kDa).
Use a concentration of 5 µg/ml. Perform heat mediated antigen retrieval before commencing with IHC staining protocol.
Use at an assay dependent concentration.
We believe this antibody is probably specific for CHFR because in IF with this antibody we see nuclear dots in SAOS2 cells (which have wt CHFR) but not in U2OS cells (that have a mutation in CHFR and hence low levels of it).
E3 ubiquitin-protein ligase that functions in the antephase checkpoint by actively delaying passage into mitosis in response to microtubule poisons. Acts in early prophase before chromosome condensation, when the centrosome move apart from each other along the periphery of the nucleus. Probably involved in signaling the presence of mitotic stress caused by microtubule poisons by mediating the 'Lys-48'-linked ubiquitination of target proteins, leading to their degradation by the proteasome. Promotes the ubiquitination and subsequent degradation of AURKA and PLK1. Probably acts as a tumor suppressor, possibly by mediating the polyubiquitination of HDAC1, leading to its degradation. May also promote the formation of 'Lys-63'-linked polyubiquitin chains and functions with the specific ubiquitin-conjugating UBC13-MMS2 (UBE2N-UBE2V2) heterodimer. Substrates that are polyubiquitinated at 'Lys-63' are usually not targeted for degradation, but are rather involved in signaling cellular stress.
Protein modification; protein ubiquitination.
Belongs to the CHFR family. Contains 1 FHA domain. Contains 1 PBZ-type zinc finger. Contains 1 RING-type zinc finger.
Weakly expressed in G1 phase, and highly expressed during S phase.
The PBZ-type zinc finger (also named CYR) mediates non-covalent poly(ADP-ribose)-binding. Poly(ADP-ribose)-binding is dependent on the presence of zinc and is required for its function in antephase checkpoint. The FHA domain plays a key role in the anti-proliferative properties of the protein and is involved in initiating a cell cycle arrest at G2/M. The FHA domain may be required to interact with phosphorylated proteins.
Poly-ADP-ribosylated. In addition to binding non covalently poly(ADP-ribose) via its PBZ-type zinc finger, the protein is also covalently poly-ADP-ribosylated by PARP1. Autoubiquitinated; may regulate its cellular level. Phosphorylated upon DNA damage, probably by ATM or ATR (By similarity). Phosphorylated by PKB. Phosphorylation may affect its E3 ligase activity.
IHC image of ab4184 staining in Human Breast adenocarcinoma 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 ab4184, 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.