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 69 kDa (predicted molecular weight: 69 kDa).
Use at an assay dependent concentration.
Use a concentration of 5 µg/ml.
Scc4 (Mau2) is the metazoan homologue of Scc4 in S. cerevisiae. In yeast, Scc4 binds to Scc2 to form an essential complex that loads cohesion onto chromosomes. Similarly, the Scc4 / Mau2 protein in metazoans plays a significant role in sister chromatid separation and in loading of cohesin onto chromatin. The Scc2-Scc4 complex is tethered to pre-replication complexes through the Cdc7-Drf1 protein kinase DDK.
ICC/IF image of ab46906 stained HeLa cells. The cells were 100% methanol fixed (5 min) then permeabilised using 0.1% PBS-Triton and then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1h to further permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody ab46906 at 1µg/ml overnight at +4°C. The secondary antibody (pseudo-colored green) was Alexa Fluor® 488 goat anti- rabbit (ab150081) IgG (H+L) preadsorbed, used at a 1/1000 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (pseudo-colored red) at a 1/200 dilution for 1h at room temperature. DAPI was used to stain the cell nuclei (pseudo-colored blue) at a concentration of 1.43µM for 1hour at room temperature.
Western blot - Scc4 antibody (ab46906)
All lanes : Anti-Scc4 antibody (ab46906) at 1 µg/ml
Lane 1 : HeLa (Human epithelial carcinoma cell line) Whole Cell Lysate Lane 2 : HeLa (Human epithelial carcinoma cell line) Nuclear Lysate
Lysates/proteins at 30 µg per lane.
Secondary Goat polyclonal to Rabbit IgG - H&L - Pre-Adsorbed (HRP) at 1/3000 dilution
Performed under reducing conditions.
Predicted band size : 69 kDa Observed band size : 69 kDa Additional bands at : 48 kDa,80-100 kDa. We are unsure as to the identity of these extra bands.
Scc4 was immunoprecipitated using 0.5mg Hela whole cell extract, 5µg of Rabbit polyclonal to Scc4 and 50µl of protein G magnetic beads (+). No antibody was added to the control (-). The antibody was incubated under agitation with Protein G beads for 10min, Hela whole cell extract lysate diluted in RIPA buffer was added to each sample and incubated for a further 10min under agitation. Proteins were eluted by addition of 40µl SDS loading buffer and incubated for 10min at 70oC; 10µl of each sample was separated on a SDS PAGE gel, transferred to a nitrocellulose membrane, blocked with 5% BSA and probed with ab46906. Secondary: Mouse monoclonal [SB62a] Secondary Antibody to Rabbit IgG light chain (HRP) (ab99697). Band: 69kDa: Scc4; non specific - 53kDa: We are unsure as to the identity of this extra band.
Western blot - Scc4 antibody (ab46906)Image kindly provided by Vlad Seitan, Institute of Human Genetics, Newcastle, United Kingdom
Lanes 1 - 2 : Blank Lanes 3 - 4 : Anti-Scc4 antibody (ab46906) at 1 µg/ml Lane 5 : Custom-made antibody at 1 µg/ml Lane 6 : Custom-made antibody at 1 µg/ml Lanes 7 - 8 : Secondary antibody only
Lane 1 : HeLa cell lysate (40 µg) Lane 2 : HeLa cell lysate (20 µg) Lane 3 : HeLa cell lysate (40 µg) Lane 4 : HeLa cell lysate (20 µg) Lane 5 : HeLa cell lysate (40 µg) Lane 6 : HeLa cell lysate (20 µg) Lane 7 : HeLa cell lysate (40 µg) Lane 8 : HeLa cell lysate (20 µg)
Developed using the ECL technique
Performed under reducing conditions.
Predicted band size : 69 kDa Observed band size : 69 kDa
Exposure time : 15 seconds
Image kindly provided by Vlad Seitan, Institute of Human Genetics, Newcastle, United Kingdom
Western blot showing detection of Scc4 in HeLa cell lysate by ab46906. Bands marked * correspond to the expected band for Scc4. The custom-made antibody was characterised by Vlad Seitan and co-workers.
Izumi K et al. Germline gain-of-function mutations in AFF4 cause a developmental syndrome functionally linking the super elongation complex and cohesin. Nat Genet47:338-44 (2015).
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