All lanes : Anti-TSG101 antibody (ab30871) at 1 µg/mlLane 1 : HeLa (Human epithelial carcinoma cell line) Whole Cell LysateLane 2 : A431 (Human epithelial carcinoma cell line) Whole Cell Lysate Lane 3 : Jurkat (Human T cell lymphoblast-like cell line) Whole Cell Lysate Lane 4 : HEK293 Human embryonic kidney cell line Whole Cell Lysate Lane 5 : HepG2 (Human hepatocellular liver carcinoma cell line) Whole Cell Lysate Lane 6 : MCF7 (Human breast adenocarcinoma cell line) Whole Cell Lysate Lane 7 : SHSY-5Y (Human neuroblastoma cell line) Whole Cell Lysate Lane 8 : U2OS (Human osteosarcoma cell line) Whole Cell LysateLysates/proteins at 10 µg per lane.SecondaryIRDye 680 Conjugated Goat Anti-Rabbit IgG (H+L) at 1/10000 dilutionPerformed under reducing conditions.
All lanes : Anti-TSG101 antibody (ab30871) at 1 µg/mlLane 1 : NIH 3T3 (Mouse embryonic fibroblast cell line) Whole Cell LysateLane 2 : PC12 (Rat adrenal pheochromocytoma cell line) Whole Cell LysateLysates/proteins at 10 µg per lane.SecondaryGoat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/10000 dilutiondeveloped using the ECL techniquePerformed under reducing conditions.
ICC/IF image of ab30871 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 (ab30871, 5µg/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 HeLa, HepG2 and MCF7 cells.
IHC image of ab30871 staining in human placenta 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 ab30871, 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.