Anti-AIF/AIFM1 Antibody Picoband® (monoclonal, 2I5)

Western blot analysis of AIF using anti-AIF antibody (M01571-1).
Electrophoresis was performed on a 5-20% SDS-PAGE gel at 70V (Stacking gel) / 90V (Resolving gel) for 2-3 hours. The sample well of each lane was loaded with 50ug of sample under reducing conditions.
Lane 1: A549 whole cell lysates,
Lane 2: Raji whole cell lysates,
Lane 3: PC-3 whole cell lysates,
Lane 4: Hela whole cell lysates,
Lane 5: Caco-2 whole cell lysates,
Lane 6: HepG2 whole cell lysates,
Lane 7: THP-1 whole cell lysates,
Lane 8: PANC-1 whole cell lysates.
After Electrophoresis, proteins were transferred to a Nitrocellulose membrane at 150mA for 50-90 minutes. Blocked the membrane with 5% Non-fat Milk/ TBS for 1.5 hour at RT. The membrane was incubated with mouse anti-AIF antigen affinity purified monoclonal antibody (Catalog # M01571-1) at 0.5 μg/mL overnight at 4°C, then washed with TBS-0.1%Tween 3 times with 5 minutes each and probed with a goat anti-mouse IgG-HRP secondary antibody at a dilution of 1:5000 for 1.5 hour at RT. The signal is developed using an Enhanced Chemiluminescent detection (ECL) kit (Catalog # EK1001) with Tanon 5200 system. A specific band was detected for AIF at approximately 70KD. The expected band size for AIF is at 70KD.

HOXA5 overexpression attenuates the malignant behaviors of ECCA cells. A , B RT-qPCR and Western blot analyses validated HOXA5 overexpression, which reduced the mitosis-related gene expression in ECCA cells. HOXA5 overexpression did not alter the EMT-related gene expression, but increased AIFM1, and decreased BCL-2 expression in ECCA cells. C HOXA5 overexpression induced cell cycle arrest in G1/G0 phase. D , E HOXA5 overexpression inhibited the proliferation of ECCA cells. F HOXA5 overexpression decreased the tumor volumes in mice. G Immunohistochemistry analysis of HOXA5 and Ki67 expression in xenograft tumors from nude mice. Data are representative images from three separate experiments. * P < 0.05. ** P < 0.01. *** P < 0.001. Orange bar, 50 μm. Green bar, 20 μm.
Index in PubMed under a CC BY license. PMID: 36167790

MXD1 overexpression reduces the malignant behaviors of ECCA cells. A , B RT-qPCR and Western blot validated MXD1 overexpression in ECCA cells. B Western blot analysis of the expression of mitosis-related genes in ECCA cells. MXD1 overexpression failed to modulate not only the EMT-related gene expression, but also the expression of AIFM1 and BCL-2 in ECCA cells. C – E MXD1 overexpression attenuated the cell cycle C , proliferation D , E of ECCA cells. F MXD1 overexpression decreased the tumor volumes in mice. G Immunohistochemistry analysis of MXD1 and Ki67 expression in the xenograft tumors from nude mice. Data are representative images of each group from three separate experiments. * P < 0.05. ** P < 0.01. *** P < 0.001. Orange bar, 50 μm. Green bar, 20 μm.
Index in PubMed under a CC BY license. PMID: 36167790

Western blot analysis of AIF using anti-AIF antibody (M01571-1).
Electrophoresis was performed on a 5-20% SDS-PAGE gel at 70V (Stacking gel) / 90V (Resolving gel) for 2-3 hours. The sample well of each lane was loaded with 50ug of sample under reducing conditions.
Lane 1: rat kidney tissue lysates,
Lane 2: rat liver tissue lysates,
Lane 3: mouse spleen lysates,
Lane 4: mouse testicular tissue lysates,
Lane 5: mouse kidney tissue lysates,
Lane 6: RAW264.7 whole cell lysates.
After Electrophoresis, proteins were transferred to a Nitrocellulose membrane at 150mA for 50-90 minutes. Blocked the membrane with 5% Non-fat Milk/ TBS for 1.5 hour at RT. The membrane was incubated with mouse anti-AIF antigen affinity purified monoclonal antibody (Catalog # M01571-1) at 0.5 μg/mL overnight at 4°C, then washed with TBS-0.1%Tween 3 times with 5 minutes each and probed with a goat anti-mouse IgG-HRP secondary antibody at a dilution of 1:5000 for 1.5 hour at RT. The signal is developed using an Enhanced Chemiluminescent detection (ECL) kit (Catalog # EK1001) with Tanon 5200 system. A specific band was detected for AIF at approximately 70KD. The expected band size for AIF is at 70KD.

IHC analysis of AIF using anti-AIF antibody (M01571-1).
AIF was detected in paraffin-embedded section of human mammary cancer tissue. Heat mediated antigen retrieval was performed in EDTA buffer (pH8.0, epitope retrieval solution). The tissue section was blocked with 10% goat serum. The tissue section was then incubated with 1μg/ml mouse anti-AIF Antibody (M01571-1) overnight at 4°C. Biotinylated goat anti-mouse IgG was used as secondary antibody and incubated for 30 minutes at 37°C. The tissue section was developed using Strepavidin-Biotin-Complex (SABC) (Catalog # SA1021) with DAB as the chromogen.

IHC analysis of AIF using anti-AIF antibody (M01571-1).
AIF was detected in paraffin-embedded section of human colon cancer tissue. Heat mediated antigen retrieval was performed in EDTA buffer (pH8.0, epitope retrieval solution). The tissue section was blocked with 10% goat serum. The tissue section was then incubated with 1μg/ml mouse anti-AIF Antibody (M01571-1) overnight at 4°C. Biotinylated goat anti-mouse IgG was used as secondary antibody and incubated for 30 minutes at 37°C. The tissue section was developed using Strepavidin-Biotin-Complex (SABC) (Catalog # SA1021) with DAB as the chromogen.

IHC analysis of AIF using anti-AIF antibody (M01571-1).
AIF was detected in paraffin-embedded section of human lung cancer tissue. Heat mediated antigen retrieval was performed in EDTA buffer (pH8.0, epitope retrieval solution). The tissue section was blocked with 10% goat serum. The tissue section was then incubated with 1μg/ml mouse anti-AIF Antibody (M01571-1) overnight at 4°C. Biotinylated goat anti-mouse IgG was used as secondary antibody and incubated for 30 minutes at 37°C. The tissue section was developed using Strepavidin-Biotin-Complex (SABC) (Catalog # SA1021) with DAB as the chromogen.

IF analysis of AIF using anti-AIF antibody (M01571-1).
AIF was detected in immunocytochemical section of MCF-7 cells. Enzyme antigen retrieval was performed using IHC enzyme antigen retrieval reagent (AR0022) for 15 mins. The cells were blocked with 10% goat serum. And then incubated with 5μg/mL mouse anti-AIF Antibody (M01571-1) overnight at 4°C. DyLight®488 Conjugated Goat Anti-Mouse IgG (BA1126) was used as secondary antibody at 1:100 dilution and incubated for 30 minutes at 37°C. The section was counterstained with DAPI. Visualize using a fluorescence microscope and filter sets appropriate for the label used.

IF analysis of AIF using anti-AIF antibody (M01571-1).
AIF was detected in paraffin-embedded section of human intestinal cancer tissue. Heat mediated antigen retrieval was performed in EDTA buffer (pH8.0, epitope retrieval solution). The tissue section was blocked with 10% goat serum. The tissue section was then incubated with 2μg/mL mouse anti-AIF Antibody (M01571-1) overnight at 4°C. DyLight®550 Conjugated Goat Anti-Mouse IgG (BA1133) was used as secondary antibody at 1:100 dilution and incubated for 30 minutes at 37°C. The section was counterstained with DAPI. Visualize using a fluorescence microscope and filter sets appropriate for the label used.

Flow Cytometry analysis of Raji cells using anti-AIF antibody (M01571-1).
Overlay histogram showing Raji cells stained with M01571-1 (Blue line). To facilitate intracellular staining, cells were fixed with 4% paraformaldehyde and permeabilized with permeabilization buffer. The cells were blocked with 10% normal goat serum. And then incubated with mouse anti-AIF Antibody (M01571-1,1μg/1x10⁶ cells) for 30 min at 20°C. DyLight®488 conjugated goat anti-mouse IgG (BA1126, 5-10μg/1x10⁶ cells) was used as secondary antibody for 30 minutes at 20°C. Isotype control antibody (Green line) was mouse IgG (1μg/1x10⁶) used under the same conditions. Unlabelled sample without incubation with primary antibody and secondary antibody (Red line) was used as a blank control.