The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
Use a concentration of 1 µg/ml. Perform heat mediated antigen retrieval before commencing with IHC staining protocol.
1/1000 - 1/10000. Detects a band of approximately 88 kDa (predicted molecular weight: 88 kDa).
Use at 2-5 µg/mg of lysate.
Core component of multiple cullin-RING-based E3 ubiquitin-protein ligase complexes which mediate the ubiquitination and subsequent proteasomal degradation of target proteins. As a scaffold protein may contribute to catalysis through positioning of the substrate and the ubiquitin-conjugating enzyme. The E3 ubiquitin-protein ligase activity of the complex is dependent on the neddylation of the cullin subunit and is inhibited by the association of the deneddylated cullin subunit with TIP120A/CAND1. The functional specificity of the E3 ubiquitin-protein ligase complex depends on the variable substrate recognition component. DCX(DET1-COP1) directs ubiquitination of JUN. DCX(DDB2) directs ubiquitination of XPC. In association with RBX1, DDB1 and DDB2 is required for histone H3 and histone H4 ubiquitination in response to ultraviolet and may be important for subsequent DNA repair. DCX(DTL) plays a role in PCNA-dependent polyubiquitination of CDT1 and MDM2-dependent ubiquitination of TP53 in response to radiation-induced DNA damage and during DNA replication. In association with DDB1 and SKP2 probably is involved in ubiquitination of CDKN1B/p27kip. Is involved in ubiquitination of HOXA9.
Protein modification; protein ubiquitination.
Belongs to the cullin family.
Neddylated. Deneddylated via its interaction with the COP9 signalosome (CSN) complex (By similarity). Deneddylated by Epstein-Barr virus BPLF1 leading to a S-phase-like environment that is required for efficient replication of the viral genome.
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human breast carcinoma (left) and mouse squamous cell carcinoma (right) tissues labelling Cullin 4a with ab72548 at 1/1000 (1µg/ml). Detection: DAB.
Immunocytochemistry/ Immunofluorescence - Anti-Cullin 4a antibody (ab72548)This image is courtesy of an anonymous Abreview
ab72548 staining Cullin 4a in human primary fibroblasts by ICC/IF (Immunocytochemistry/immunofluorescence). Cells were fixed with paraformaldehyde and blocked with 2% BSA for 2 hours at room temperature. Samples were incubated with primary antibody (1/300 in PBS + 2% BSA) for 14 hours at 4°C. An Alexa Fluor® 488-conjugated goat anti-rabbit IgG polyclonal (13200) was used as the secondary antibody.
All lanes : Anti-Cullin 4a antibody (ab72548) at 0.1 µg/ml
Lane 1 : HeLa whole cell lysate at 50 µg Lane 2 : HeLa whole cell lysate at 15 µg Lane 3 : HeLa whole cell lysate at 5 µg Lane 4 : 293T whole cell lysate at 50 µg Lane 5 : NIH3T3 whole cell lysate at 50 µg
Predicted band size: 88 kDa Observed band size: 88 kDa Additional bands at: 117 kDa. We are unsure as to the identity of these extra bands.
Detection of Human Cullin 4a by Immunoprecipitation from Whole cell lysate from HeLa cells (1 mg for IP, 20% of IP loaded), using ab72548 at 3 µg/mg lysate for IP and at 1 µg/ml for subsequent Western blot detection.
IHC image of ab72548 staining in human normal cervix 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 EDTA (pH9, epitope retrieval solution 2) for 20 mins. The section was then incubated with ab72548, 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.