Anti-MSI2 抗体 [EP1305Y] (ab76148)

Lanes 1 - 4: Merged signal (red and green). Green - ab76148 observed at 40 kDa. Red - loading control, ab18058, observed at 130 kDa.

ab76148 was shown to specifically react with MSI2 in wild-type HAP1 cells as signal was lost in MSI2 knockout cells. Wild-type and MSI2 knockout samples were subjected to SDS-PAGE. Ab76148 and ab18058 (Mouse anti-Vinculin loading control) were incubated overnight at 4°C at 1/1000 dilution and 1/20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preabsorbed ab216773 and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed ab216776 secondary antibodies at 1/20000 dilution for 1 hour at room temperature before imaging.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of Human bladder carcinoma tissue sections labeling MSI2 with purified ab76148 at 1:500 dilution (2.14 µg/ml). Heat mediated antigen retrieval was performed using ab93684 (Tris/EDTA buffer, pH 9.0). ImmunoHistoProbe one step HRP Polymer (ready to use)was used as the secondary antibody. Negative control:PBS instead of the primary antibody. Hematoxylin was used as a counterstain.

Intracellular Flow Cytometry analysis of T-47D (Human ductal breast epithelial tumor epithelial cell) cells labeling MSI2 with purified ab76148 at 1/100 dilution (10 µg/ml) (red). Cells were fixed with 4% Paraformaldehyde. A Goat anti rabbit IgG (Alexa Fluor^® 488, ab150077) secondary antibody was used at 1/2000. Isotype control - Rabbit monoclonal IgG (Black). Unlabeled control - Cell without incubation with primary antibody and secondary antibody (Blue).

Immunohistochemical analysis of paraffin-embedded human placenta with ab76148 at 1/100-1/250 dilution.

Heat mediated antigen retrieval was performed via the pressure cooker method before commencing with IHC staining protocol.

Blocking and diluting buffer and concentration: 5% NFDM/TBST.

Overlay histogram showing HeLa cells stained with ab76148 (red line). The cells were fixed with 4% paraformaldehyde (10 min) and then permeabilized with 0.1% PBS-Tween for 20 min. The cells were then incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions followed by the antibody (ab76148, 1/100 dilution) for 30 min at 22°C. The secondary antibody used was DyLight® 488 goat anti-rabbit IgG (H+L) (ab96899) at 1/500 dilution for 30 min at 22°C. Isotype control antibody (black line) was rabbit IgG (monclonal) (1µg/1x10⁶ cells) used under the same conditions. Acquisition of >5,000 events was performed. This antibody gave a positive result in HeLa cells fixed with 80% methanol (5 min)/permeabilized in 0.1% PBS-Tween for 20 min used under the same conditions.