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Recombinant fragment, corresponding to amino acids 1-202 of Mouse Bmi1
Our Abpromise guarantee covers the use of ab14389 in the following tested applications.
The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
|ICC/IF||Use a concentration of 0.65 µg/ml. PubMed: 19001505|
|Flow Cyt||Use a concentration of 0.65 µg/ml. PubMed: 19001505
ab170190 - Mouse monoclonal IgG1, is suitable for use as an isotype control with this antibody.
|ChIP||Use at an assay dependent concentration. PubMed: 18332116|
|WB||Use a concentration of 0.2 - 2 µg/ml. Predicted molecular weight: 37 kDa. Detects Bmi-1 in RIPA lysates from U2OS cells. U2OS cell lysate was resolved by electrophoresis, transferred to nitrocellulose and probed with ab14389 0.2ug/ml. Proteins were visualized using a goat anti-mouse IgG labeled with HRP and a chemiluminescence detection system.|
|ICC||Use at an assay dependent concentration.|
|IP||Use at an assay dependent concentration.|
|IHC-Fr||Use at an assay dependent concentration. PubMed: 17210912|
Lane 1: Wild-type HAP1 cell lysate (20 µg)
Lane 2: Bmi1 knockout HAP1 cell lysate)
Lane 3: U2OS cell lysate (40 ug)
Lane 4: Molt-4 cell lysate (40ug)
Lanes 1 - 4: Merged signal (red and green). Green - ab14389 observed at 42 kDa. Red - loading control, ab176560, observed at 52 kDa.
ab14389 was shown to recognize Bmi1 when Bmi1 knockout samples were used, along with additional cross-reactive bands. Wild-type and Bmi1 knockout samples were subjected to SDS-PAGE. ab14389 at a concentration of 1 μg/ml and ab176560 (loading control to alpha tubulin) diluted to 1/10000 were incubated overnight at 4°C. Blots were developed with Goat anti-Mouse IgG H&L (IRDye® 800CW) preadsorbed (ab216772) and Goat Anti-Rabbit IgG H&L (IRDye® 680RD) preadsorbed (ab216777) secondary antibodies at 1/10000 dilution for 1 hour at room temperature before imaging.
SK-N-SH cells were fixed in 4% paraformaldehyde, permeabilized in 0.55 Triton-X100 and incubated for 1 hour with ab14389 (1/200). The Bmi1 staining is shown in red. The cells were counterstained with DAPI (blue). 100x magnification.
Cells were lysed in RIPA buffer. Samples (25 µg) of total protein were separated by SDS-polyacrylamide gel electrophoresis gel (PAGE) in a 12% gel and transferred onto a nitrocellulose membrane. This was followed by an incubation with mouse anti-BMI1 (ab14389) and a sheep anti-mouse HRP conjugated antibody diluted 1:2000 was used as a secondary antibody. HRP conjugated anti-GAPDH antibody (ab9482) was used as a loading control.
The mCbx7 expressing cells were infected with lentivirus-based shRNA vectors targeting BMI1 or an irrelevant control (C).
Formalin-fixed paraffin-embedded (FFPE) colorectal cancer tissues and normal colorectal tissues. Endogenous peroxidase was blocked by incubating the sections in a 0.3% solution of hydrogen peroxide (in PBS) for 20 min. Antigen retrieval was performed by heating the sections for 10 min at 95°C in a citrate buffer. Tissues were incubated with ab14389 overnight (16 hrs). Staining was visualized using the Dako REAL EnVision Detection System, Peroxidase/DAB+, Rabbit/Mouse (Dako). The stained TMA sections were scanned using a 20x magnification on the semi-automated Ariol system (Leica Microsystems, Wetzlar, Germany). Tumor cell areas (tumor tissues) and colon epithelium (in normal tissues) were identified for positive (indicated by yellow dots) and negative (blue dots) nuclei in tumor cores. TMA slides were scanned using a 20x magnification. Shown are positively stained tumor cores (top image) and negative tumor cores (bottom image).
Western blot analysis of RIPA lysates from U2OS cells labelling Bmi-1 with ab14389 at 0.2μg . An IgG (HRP) goat anti-mouse was used as the secondary antibody.
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