Anti-IMP3 antibody [EPR12021]

• Flow Cyt (Intra)

Lab

Flow Cytometry (Intracellular) - Anti-IMP3 antibody [EPR12021] (AB177477)

Intracellular Flow Cytometry analysis of BxPC-3 (human pancreas adenocarcinoma) cells labeling with purified ab177477 at 1/300 dilution (10ug/ml) (Red). Cells were fixed with 80% methanoland permeabilised with 0.1% Tween-20. A Goat anti rabbit IgG (Alexa Fluor^® 488) (ab150077) (1/2000 dilution) was used as the secondary antibody. Rabbit monoclonal IgG (Black) (ab172730) was used as a isotype control. Cell without incubation with primary antibody and secondary antibody (Blue) were used as unlabeled control.

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-IMP3 antibody [EPR12021] (AB177477)

Immunocytochemistry/ Immunofluorescence analysis of HeLa (Human cervix adenocarcinoma epithelial cell) cells labeling IMP3 using ab177477. The cells were fixed with 4% paraformaldehyde then permeabilized with 0.1% Triton X-100. The cells were then incubated with ab177477 at 1 : 50 dilution followed by a further incubation with a Goat anti rabbit IgG (Alexa Fluor® 488, ab150077) at 2 μg/ml (shown in green). Nuclear DNA was labelled in blue with DAPI. Cells were counterstained using ab195889 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor® 594) at 1 : 200 dilution (shown in red). Secondary antibody only control : PBS instead of the primary antibody.

• IP

Lab

Immunoprecipitation - Anti-IMP3 antibody [EPR12021] (AB177477)

Immunoprecipitation of IMP3 from 293T (Human embryonic kidney epithelial cell) whole cell lysate using ab177477 at 1/20 dilution. Lane 1 : 293T (Human embryonic kidney epithelial cell) whole cell lysate 10μg Lane 2 : 293T whole cell lysate Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab177477 in 293T whole cell lysate ab131366 was used as secondary antibody at 1/1000 dilution. Blocking and diluting buffer and concentration : 5% NFDM/TBST. Observed MW at 69kDa.

All lanes:

Immunoprecipitation - Anti-IMP3 antibody [EPR12021] (ab177477) at 1/20 dilution

Lane 1:

293T (Human embryonic kidney epithelial cell) whole cell lysate at 10 µg

Lane 2:

293T whole cell lysate

Lane 3:

Rabbit monoclonal IgG (<a href='/en-us/products/primary-antibodies/rabbit-igg-monoclonal-epr25a-isotype-control-ab172730'>ab172730</a>) instead of ab177477 in 293T whole cell lysate

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 1/1000 dilution

Predicted band size: 63 kDa

Observed band size: 69 kDa

false

• WB

Lab

Western blot - Anti-IMP3 antibody [EPR12021] (AB177477)

Lane 1 : Wild-type HAP1 whole cell lysate (20 μg)
Lane 2 : IGF2BP3 (IMP3) knockout HAP1 whole cell lysate (20 μg)
Lane 3 : HeLa whole cell lysate (20 μg)
Lane 4 : HEK293 whole cell lysate (20 μg)

Lanes 1 - 4 : Merged signal (red and green). Green - ab177477 observed at 70 kDa. Red - loading control, ab9484, observed at 37 kDa.

ab177477 was shown to specifically react with IGF2BP3 (IMP3) in wild-type HAP1 cells along with additional cross-reactive bands. No band was observed when IGF2BP3 (IMP3) knockout cells were examined. Wild-type and IGF2BP3 (IMP3) knockout samples were subjected to SDS-PAGE. ab177477 and ab9484 (Mouse anti GAPDH loading control) were incubated overnight at 4°C at 1/1000 dilution and 1/20,000 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 1/20,000 dilution for 1 hour at room temperature before imaging.

All lanes:

Western blot - Anti-IMP3 antibody [EPR12021] (ab177477)

Predicted band size: 63 kDa

false

• WB

Lab

Western blot - Anti-IMP3 antibody [EPR12021] (AB177477)

All lanes:

Western blot - Anti-IMP3 antibody [EPR12021] (ab177477) at 1/10000 dilution

Lane 1:

MDA-MB-231 (Human breast adenocarcinoma epithelial cell) whole cell lysate at 20 µg

Lane 2:

MDA-MB-435S (Human mammary gland ductal carcinoma melanocyte) whole cell lysate at 20 µg

Lane 3:

HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysate at 20 µg

Lane 4:

HEK-293 (Human embryonic kidney epithelial cell) whole cell lysate at 20 µg

Lane 5:

Mouse placenta lysate at 20 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: 63 kDa

Observed band size: 69 kDa

false

• WB

Lab

Western blot - Anti-IMP3 antibody [EPR12021] (AB177477)

Blocking and diluting buffer and concentration : 5% NFDM/TBST. We are unsure about the nature of the extra bands.

All lanes:

Western blot - Anti-IMP3 antibody [EPR12021] (ab177477) at 1/10000 dilution

Lane 1:

Mouse spleen lysate at 20 µg

Lane 2:

Rat heart lysate at 20 µg

Lane 3:

Rat spleen lysate at 20 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: 63 kDa

Observed band size: 69 kDa

false