Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human colon carcinoma tissue labelling SPTBN1 with ab72239 at 1/1000 (0.2µg/ml). Detection: DAB.
Western blot - Anti-SPTBN1 antibody (ab72239)
All lanes : Anti-SPTBN1 antibody (ab72239) at 0.04 µg/ml
Lane 1 : Whole cell lysate from Hela cells at 50 µg Lane 2 : Whole cell lysate from Hela cells at 15 µg Lane 3 : Whole cell lysate from Hela cells at 5 µg Lane 4 : Whole cell lysate from 293T cells at 50 µg
Predicted band size: 274 kDa Observed band size: 274 kDa
Immunoprecipitation/ Western Blot of SPTBN1.
Lane 1: ab72239 at 3µg/mg whole cell lysate.
Lane 2: Control IgG.
ab72239 at 1µg/ml for WB.
Whole cell lysate from Hela cells at 1mg for IP, 20% of IP loaded.
Chemiluminescence with an exposure time of 1 second.
IHC image of ab72239 staining in human normal tonsil 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 ab72239, 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.
ICC/IF image of ab72239 stained A431 cells. The cells were 100% methanol fixed (10 min) and then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1h to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab72239, 1µg/ml) overnight at +4°C. The secondary antibody (green) was ab96899, DyLight® 488 goat anti-rabbit IgG (H+L) used at a 1/250 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (red) at a 1/200 dilution for 1h. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43µM.
Kiriyama S et al. CEACAM1 long cytoplasmic domain isoform is associated with invasion and recurrence of hepatocellular carcinoma. Ann Surg Oncol21 Suppl 4:S505-14 (2014).
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