All lanes : Anti-USP22 antibody (ab4812) at 1 µg/mlLane 1 : HeLa (Human epithelial carcinoma cell line) Whole Cell Lysate Lane 2 : HeLa (Human epithelial carcinoma cell line) Nuclear Lysate Lane 3 : Jurkat (Human T cell lymphoblast-like cell line) Whole Cell Lysate Lane 4 : Jurkat (Human T cell lymphoblast-like cell line) Nuclear Lysate (ab14844)Lane 5 : HepG2 (Human hepatocellular liver carcinoma cell line) Whole Cell Lysate Lane 6 : HepG2 (Human hepatocellular liver carcinoma cell line) Nuclear Lysate (ab14660)Lane 7 : A431 (Human epithelial carcinoma cell line) Whole Cell Lysate Lysates/proteins at 10 µg per lane.SecondaryIRDye 680 Conjugated Goat Anti-Rabbit IgG (H+L) at 1/10000 dilutionPerformed under reducing conditions.
ICC/IF image of ab4812 stained human HEK 293 cells. The cells were 4% PFA fixed (10 min), permabilised in 0.1% PBS-Tween (20 min) and incubated with the antibody (ab4812, 1µg/ml) for 1h at room temperature. 1%BSA / 10% normal goat serum / 0.3M glycine was used to 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). This antibody also gave a positive IF result in HepG2 and MCF7 cells.
ab4812 (1/200) staining USP22 in assynchronous HeLa cells (green). Cells were fixed in paraformaldehyde, permeabilized in 0.5% Triton X-100 and counterstained with DAPI in order to highlight the nucleus (red). For further experimental details please refer to Abreview.See Abreview
IHC image of ab4812 staining in human heart 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 ab4812, 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.
USP22 was immunoprecipitated using 0.5mg Hela whole cell extract, 5µg of Rabbit polyclonal to USP22 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 ab4812.Secondary: Mouse monoclonal [SB62a] Secondary Antibody to Rabbit IgG light chain (HRP) (ab99697).Band: 66kDa: USP22; non specific - 75kDa: We are unsure as to the identity of this extra band.