AIF/AIFM1 Antibody (monoclonal, 2I5)

• Catalog Number: orb547790
• Category: Antibodies
• Description: AIF/AIFM1 Antibody (monoclonal, 2I5)
• Species/Host: Mouse
• Clonality: Monoclonal
• Clone Number: 2I5
• Tested applications: FC, ICC, IF, IHC, WB
• Reactivity: Human, Mouse, Rat
• Isotype: Mouse IgG1
• Immunogen: A synthetic peptide corresponding to a sequence at the C-terminus of human AIF (582-613aa FNRMPIARKIIKDGEQHEDLNEVAKLFNIHED), identical to the related mouse and rat sequences.
• Concentration: Adding 0.2 ml of distilled water will yield a concentration of 500 μg/ml.
• Dilution range: Western blot, 0.1-0.5μg/ml Immunohistochemistry (Paraffin-embedded Section), 0.5-1μg/ml Immunofluorescence, 2μg/ml Immunocytochemistry/Immunofluorescence, 5μg/ml Flow Cytometry, 1-3μg/1x106 cells
• Form/Appearance: Lyophilized
• Conjugation: Unconjugated
• MW: 70 kDa
• UniProt ID: O95831
• Storage: Store at -20˚C for one year from date of receipt. After reconstitution, at 4˚C for one month. It can also be aliquotted and stored frozen at -20˚C for six months. Avoid repeated freeze-thaw cycles.
• Alternative names: AIFM1 antibody|AIFM1_HUMAN antibody|Apoptosis indu
• Note: For research use only
• Application notes: Add 0.2ml of distilled water will yield a concentration of 500μg/ml.
• Expiration Date: 12 months from date of receipt.

Western blot analysis of AIF using anti-AIF antibody (orb547790). 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 # orb547790) 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 # orb90502) with Tanon 5200 system. A specific band was detected for AIF at approximately 70KD. The expected band size for AIF is at 70KD.

Western blot analysis of AIF using anti-AIF antibody (orb547790). 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 # orb547790) 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 # orb90502) 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 (orb547790). 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 (orb547790) 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 # orb90443) with DAB as the chromogen.

IHC analysis of AIF using anti-AIF antibody (orb547790). 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 (orb547790) 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 # orb90443) with DAB as the chromogen.

IHC analysis of AIF using anti-AIF antibody (orb547790). 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 (orb547790) 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 # orb90443) with DAB as the chromogen.

IF analysis of AIF using anti-AIF antibody (orb547790). AIF was detected in immunocytochemical section of MCF-7 cells. Enzyme antigen retrieval was performed using IHC enzyme antigen retrieval reagent (orb90553) for 15 mins. The cells were blocked with 10% goat serum. And then incubated with 5μg/mL mouse anti-AIF Antibody (orb547790) overnight at 4°C. DyLight®488 Conjugated Goat Anti-Mouse IgG 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 (orb547790). 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 (orb547790) overnight at 4°C. DyLight?550 Conjugated Goat Anti-Mouse IgG 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 (orb547790). Overlay histogram showing Raji cells stained with orb547790 (Blue line). The cells were blocked with 10% normal goat serum. And then incubated with mouse anti-AIF Antibody (orb547790, 1μg/1x10^6 cells) for 30 min at 20°C. DyLight®488 conjugated goat anti-mouse IgG (5-10μg/1x10^6 cells) was used as secondary antibody for 30 minutes at 20°C. Isotype control antibody (Green line) was mouse IgG (1μg/1x10^6) used under the same conditions. Unlabelled sample (Red line) was also used as a control.