Anti-MSI2 antibody [EP1305Y]

• Flow Cyt (Intra)

Unknown

Flow Cytometry (Intracellular) - Anti-MSI2 antibody [EP1305Y] (AB76148)

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.

• ICC/IF

Unknown

Immunocytochemistry/ Immunofluorescence - Anti-MSI2 antibody [EP1305Y] (AB76148)

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 Cyt (Intra)

Unknown

Flow Cytometry (Intracellular) - Anti-MSI2 antibody [EP1305Y] (AB76148)

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).

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-MSI2 antibody [EP1305Y] (AB76148)

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.

• IP

Unknown

Immunoprecipitation - Anti-MSI2 antibody [EP1305Y] (AB76148)

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

All lanes:

Immunoprecipitation - 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

Lane 2:

T-47D whole cell lysate. ab76148 use as the capture antibody at 1:50 dilution.

Lane 3:

T-47D whole cell lysate. <a href='/en-us/products/primary-antibodies/rabbit-igg-monoclonal-epr25a-isotype-control-ab172730'>ab172730</a> use as the capture antibody at 1:50 dilution.

Secondary

All lanes:

Immunoprecipitation - VeriBlot for IP Detection Reagent (HRP) (<a href='/en-us/products/reagents/veriblot-for-ip-detection-reagent-hrp-ab131366'>ab131366</a>) at 1/5000 dilution

Predicted band size: 35 kDa

Observed band size: 35 kDa

false

Exposure time: 5s

• WB

Unknown

Western blot - Anti-MSI2 antibody [EP1305Y] (AB76148)

All lanes:

Western blot - Anti-MSI2 antibody [EP1305Y] (ab76148) at 1/2000 dilution

Lane 1:

Rat brain cell lysates at 10 µg

Lane 2:

Human brain cell lysates at 10 µg

Lane 3:

SW480 cell lysates at 10 µg

Lane 4:

T47D cell lysates at 10 µg

Secondary

All lanes:

HRP labelled goat anti-rabbit at 1/2000 dilution

Predicted band size: 35 kDa

Observed band size: 35 kDa

false

• WB

Lab

Western blot - 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.

All lanes:

Western blot - Anti-MSI2 antibody [EP1305Y] (ab76148) at 1/1000 dilution

Lane 1:

Wild-type HAP1 whole cell lysate at 20 µg

Lane 2:

MSI2 knockout HAP1 whole cell lysate at 20 µg

Lane 3:

HeLa whole cell lysate at 20 µg

Lane 4:

MCF7 whole cell lysate at 20 µg

Predicted band size: 35 kDa

false

• WB

Lab

Western blot - Anti-MSI2 antibody [EP1305Y] (AB76148)

All lanes:

Western blot - Anti-MSI2 antibody [EP1305Y] (ab76148) at 1/1000 dilution

Lane 1:

T-47D (Human ductal breast epithelial tumor epithelial cell) whole cell lysates at 15 µg

Lane 2:

A549 (Human lung carcinoma epithelial cell) whole cell lysates at 15 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

Predicted band size: 35 kDa

Observed band size: 35 kDa

false

• WB

Lab

Western blot - Anti-MSI2 antibody [EP1305Y] (AB76148)

All lanes:

Western blot - Anti-MSI2 antibody [EP1305Y] (ab76148) at 1/1000 dilution

Lane 1:

Mouse brain lysates at 15 µg

Lane 2:

Rat brain lysates at 15 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

Predicted band size: 35 kDa

Observed band size: 35 kDa

false