All lanes : Anti-MSI2 antibody [EP1305Y] (ab76148) at 1/1000 dilution

Lane 1 : Wild-type HAP1 whole cell lysate
Lane 2 : MSI2 knockout HAP1 whole cell lysate
Lane 3 : HeLa whole cell lysate
Lane 4 : MCF7 whole cell lysate

Lysates/proteins at 20 µg per lane.

Predicted band size: 35 kDa

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.

All lanes : Anti-MSI2 antibody [EP1305Y] (ab76148) at 1/1000 dilution

Lane 1 : Mouse brain lysates
Lane 2 : Rat brain lysates

Lysates/proteins at 15 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution

Predicted band size: 35 kDa
Observed band size: 35 kDa

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.

All lanes : Anti-MSI2 antibody [EP1305Y] (ab76148) at 1/1000 dilution

Lane 1 : T-47D (Human ductal breast epithelial tumor epithelial cell) whole cell lysates
Lane 2 : A549 (Human lung carcinoma epithelial cell) whole cell lysates

Lysates/proteins at 15 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution

Predicted band size: 35 kDa
Observed band size: 35 kDa

Immunocytochemistry/ Immunofluorescence analysis of MCF7 (Human breast adenocarcinoma epithelial cell) cells labeling MSI2 with purified ab76148 at 1:100 dilution (10 µg/ml). Cells were fixed in 4% Paraformaldehyde and permeabilized with 0.1% tritonX-100. Cells were counterstained with None. Goat anti rabbit IgG (Alexa Fluor® 488, ab150077) was used as the secondary antibody at 1:1000 (2 µg/ml) dilution. DAPI nuclear counterstain. PBS instead of the primary antibody was used as the secondary antibody only control.

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.

All lanes : Anti-MSI2 antibody [EP1305Y] (ab76148) at 1/1000 dilution

Lane 1 : T-47D (Human ductal breast epithelial tumor epithelial cell) whole cell lysate at 10 µg with 5% NFDM/TBST
Lane 2 : T-47D whole cell lysate. ab76148 use as the capture antibody at 1:50 dilution. with 5% NFDM/TBST
Lane 3 : T-47D whole cell lysate. ab172730 use as the capture antibody at 1:50 dilution. with 5% NFDM/TBST

Secondary
All lanes : VeriBlot for IP Detection Reagent (HRP) (ab131366) at 1/5000 dilution (VeriBlot for IP secondary antibody (HRP))

Observed band size: 35 kDa why is the actual band size different from the predicted?


Exposure time: 5 seconds

All lanes : Anti-MSI2 antibody [EP1305Y] (ab76148) at 1/2000 dilution

Lane 1 : Rat brain cell lysates
Lane 2 : Human brain cell lysates
Lane 3 : SW480 cell lysates
Lane 4 : T47D cell lysates

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : HRP labelled goat anti-rabbit at 1/2000 dilution

Predicted band size: 35 kDa
Observed band size: 35 kDa

ICC/IF image of ab76148 stained PC12 cells. The cells were 4% formaldehyde 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 (ab76148, 1µg/ml) overnight at +4°C. 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) at a 1/200 dilution for 1h. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43µM.

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 22ºC. The secondary antibody used was DyLight® 488 goat anti-rabbit IgG (H+L) (ab96899) at 1/500 dilution for 30 min at 22ºC. 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.