Recombinant Anti-MSI2 antibody [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.

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.