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

Figure 1. 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.
Figure 2. 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.
Figure 3. 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 1 µg/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.
Figure 4. 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 1 µg/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.
Figure 5. 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 1 µg/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.
Figure 5. 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 5 µg/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.
Figure 6. 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 2 µg/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.
Figure 7. Flow Cytometry analysis of Raji cells using anti-AIF antibody (M01571-1).
Overlay histogram showing Raji cells stained with M01571-1 (Blue line).The cells were blocked with 10% normal goat serum. And then incubated with mouse anti-AIF Antibody (M01571-1,1 µg/1x10⁶ cells) for 30 min at 20°C. DyLight®488 conjugated goat anti-mouse IgG (BA1126, 5-10 µg/1x10⁶ cells) was used as secondary antibody for 30 minutes at 20°C. Isotype control antibody (Green line) was mouse IgG (1 µg/1x10⁶) used under the same conditions. Unlabelled sample (Red line) was also used as a control.

Figure 1. 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.

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BACKGROUND

Product Information
Application Applications Legend: WB=Western Blot IHC=Immunohistochemistry IHC-P=Immunohistochemistry (Paraffin-embedded Sections) IHC-F=Immunohistochemistry (Frozen Sections) IF=Immunofluorescence FC=Flow Cytopmetry IC=Immunochemistry ICC=Immunocytochemistry E=ELISA IP=Immunoprecipitation DB=Dot Blot CHIP=Chromatin Immunoprecipitation FA=Fluorescence Assay IEM=Immuno electron microscopy EIA=Enzyme Immunoassay	WB, IHC, IF, ICC, FC
Primary Accession	O95831
Host	Mouse
Isotype	Mouse IgG1
Reactivity	Rat, Human, Mouse
Clonality	Monoclonal
Format	Lyophilized
Description	Anti-AIF/AIFM1 Antibody Picoband™ (monoclonal, 2I5) . Tested in Flow Cytometry, IF, IHC, ICC, WB applications. This antibody reacts with Human, Mouse, Rat.
Reconstitution	Add 0.2ml of distilled water will yield a concentration of 500 µg/ml.

Additional Information
Gene ID	9131
Other Names	Apoptosis-inducing factor 1, mitochondrial, 1.6.99.-, Programmed cell death protein 8, AIFM1 (HGNC:8768), AIF, PDCD8
Calculated MW	70 kDa
Application Details	Western blot, 0.1-0.5 µg/ml Immunohistochemistry (Paraffin-embedded Section), 0.5-1 µg/ml Immunofluorescence, 2 µg/ml Immunocytochemistry/Immunofluorescence, 5 µg/ml Flow Cytometry, 1-3 µg/1x10^6 cells
Subcellular Localization	Mitochondrion intermembrane space. Mitochondrion inner membrane. Nucleus. Cytoplasm. Perinuclear region.
Tissue Specificity	Detected in muscle and skin fibroblasts (at protein level). Isoform 5 is frequently down-regulated in human cancers.
Protein Name	Apoptosis-inducing factor 1, mitochondrial
Contents	Each vial contains 4mg Trehalose, 0.9mg NaCl, 0.2mg Na2HPO4, 0.05mg NaN3.
Clone Names	Clone: 2I5
Immunogen	A synthetic peptide corresponding to a sequence at the C-terminus of human AIF, identical to the related mouse and rat sequences.
Cross Reactivity	No cross-reactivity with other proteins.
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.

Protein Information
Name	AIFM1 (HGNC:8768)
Synonyms	AIF, PDCD8
Function	Functions both as NADH oxidoreductase and as regulator of apoptosis (PubMed:17094969, PubMed:20362274, PubMed:23217327, PubMed:33168626). In response to apoptotic stimuli, it is released from the mitochondrion intermembrane space into the cytosol and to the nucleus, where it functions as a proapoptotic factor in a caspase- independent pathway (PubMed:20362274). Release into the cytoplasm is mediated upon binding to poly-ADP-ribose chains (By similarity). The soluble form (AIFsol) found in the nucleus induces 'parthanatos' i.e. caspase-independent fragmentation of chromosomal DNA (PubMed:20362274). Binds to DNA in a sequence-independent manner (PubMed:27178839). Interacts with EIF3G, and thereby inhibits the EIF3 machinery and protein synthesis, and activates caspase-7 to amplify apoptosis (PubMed:17094969). Plays a critical role in caspase-independent, pyknotic cell death in hydrogen peroxide-exposed cells (PubMed:19418225). In contrast, participates in normal mitochondrial metabolism. Plays an important role in the regulation of respiratory chain biogenesis by interacting with CHCHD4 and controlling CHCHD4 mitochondrial import (PubMed:26004228).
Cellular Location	Mitochondrion intermembrane space. Mitochondrion inner membrane. Cytoplasm. Nucleus. Cytoplasm, perinuclear region. Note=Proteolytic cleavage during or just after translocation into the mitochondrial intermembrane space (IMS) results in the formation of an inner-membrane-anchored mature form (AIFmit). During apoptosis, further proteolytic processing leads to a mature form, which is confined to the mitochondrial IMS in a soluble form (AIFsol). AIFsol is released to the cytoplasm in response to specific death signals, and translocated to the nucleus, where it induces nuclear apoptosis (PubMed:15775970). Release into the cytoplasm is mediated upon binding to poly-ADP-ribose chains (By similarity) Translocation into the nucleus is promoted by interaction with (auto- poly-ADP-ribosylated) processed form of PARP1 (PubMed:33168626) Colocalizes with EIF3G in the nucleus and perinuclear region (PubMed:17094969). {ECO:0000250\|UniProtKB:Q9Z0X1, ECO:0000269\|PubMed:15775970, ECO:0000269\|PubMed:17094969, ECO:0000269\|PubMed:33168626} [Isoform 4]: Mitochondrion. Cytoplasm, cytosol. Note=In pro-apoptotic conditions, is released from mitochondria to cytosol in a calpain/cathepsin-dependent manner.
Tissue Location	Expressed in all tested tissues (PubMed:16644725). Detected in muscle and skin fibroblasts (at protein level) (PubMed:23217327). Expressed in osteoblasts (at protein level) (PubMed:28842795). [Isoform 4]: Expressed in all tested tissues except brain.

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