Validated using a knockout cell lineRecombinantRabMAb

Recombinant Anti-AIF antibody [E20] - Mitochondrial Marker (ab32516)

Reviews (5)Q&A (3)References (59)
Western blot - Anti-AIF antibody [E20] - Mitochondrial Marker (ab32516)
  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-AIF antibody [E20] - Mitochondrial Marker (ab32516)
  • Western blot - Anti-AIF antibody [E20] - Mitochondrial Marker (ab32516)
  • Immunoprecipitation - Anti-AIF antibody [E20] - Mitochondrial Marker (ab32516)
  • Immunocytochemistry/ Immunofluorescence - Anti-AIF antibody [E20] - Mitochondrial Marker (ab32516)
  • Flow Cytometry (Intracellular) - Anti-AIF antibody [E20] - Mitochondrial Marker (ab32516)
  • Anti-AIF antibody [E20] - Mitochondrial Marker (ab32516)

Key features and details

  • Produced recombinantly (animal-free) for high batch-to-batch consistency and long term security of supply
  • Rabbit monoclonal [E20] to AIF - Mitochondrial Marker
  • Suitable for: Flow Cyt (Intra), ICC/IF, IHC-Fr, WB, IHC-P, IP
  • Knockout validated
  • Reacts with: Mouse, Rat, Human

Conjugates logo Related conjugates and formulations

Alexa Fluor® 647 Biotin Carrier Free

Overview

  • Product name

    Anti-AIF antibody [E20] - Mitochondrial Marker
    See all AIF primary antibodies

  • Description

    Rabbit monoclonal [E20] to AIF - Mitochondrial Marker

  • Host species

    Rabbit

  • Tested applications

    Suitable for: Flow Cyt (Intra), ICC/IF, IHC-Fr, WB, IHC-P, IPmore details

  • Species reactivity

    Reacts with: Mouse, Rat, Human

  • Immunogen

    Synthetic peptide. This information is proprietary to Abcam and/or its suppliers.

  • Positive control

    • WB: HEK-293T and K562 cell lysate. IHC-P: Human cervical carcinoma tissue.

  • General notes

    This product is a recombinant monoclonal antibody, which offers several advantages including:

    • - High batch-to-batch consistency and reproducibility
    • - Improved sensitivity and specificity
    • - Long-term security of supply
    • - Animal-free production
    For more information see here.

    Our RabMAb® technology is a patented hybridoma-based technology for making rabbit monoclonal antibodies. For details on our patents, please refer to RabMAb® patents.

Properties

Applications

The Abpromise guarantee

Our Abpromise guarantee covers the use of ab32516 in the following tested applications.

The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.

Application Abreviews Notes
Flow Cyt (Intra)
1/50.

ab172730 - Rabbit monoclonal IgG, is suitable for use as an isotype control with this antibody.
ICC/IF
1/500.
IHC-Fr (1)
Use at an assay dependent concentration. PubMed: 23118224
WB (3)
1/1000. Detects a band of approximately 67 kDa (predicted molecular weight: 67 kDa).
IHC-P (1)
Use at an assay dependent concentration. Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.
IP
Use at an assay dependent concentration.
Notes
Flow Cyt (Intra)
1/50.

ab172730 - Rabbit monoclonal IgG, is suitable for use as an isotype control with this antibody.
ICC/IF
1/500.
IHC-Fr
Use at an assay dependent concentration. PubMed: 23118224
WB
1/1000. Detects a band of approximately 67 kDa (predicted molecular weight: 67 kDa).
IHC-P
Use at an assay dependent concentration. Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.
IP
Use at an assay dependent concentration.

Target

  • Function

    Probable oxidoreductase that has a dual role in controlling cellular life and death; during apoptosis, it is translocated from the mitochondria to the nucleus to function as a proapoptotic factor in a caspase-independent pathway, while in normal mitochondria, it functions as an antiapoptotic factor via its oxidoreductase activity. The soluble form (AIFsol) found in the nucleus induces 'parthanatos' i.e., caspase-independent fragmentation of chromosomal DNA. Interacts with EIF3G,and thereby inhibits the EIF3 machinery and protein synthesis, and activates casapse-7 to amplify apoptosis. Plays a critical role in caspase-independent, pyknotic cell death in hydrogen peroxide-exposed cells. Binds to DNA in a sequence-independent manner.

  • Involvement in disease

    Defects in AIFM1 are the cause of combined oxidative phosphorylation deficiency type 6 (COXPD6) [MIM:300816]. It is a mitochondrial disease resulting in a neurodegenerative disorder characterized by psychomotor delay, hypotonia, areflexia, muscle weakness and wasting.

  • Sequence similarities

    Belongs to the FAD-dependent oxidoreductase family.

  • Post-translational
    modifications

    Under normal conditions, a 54-residue N-terminal segment is first proteolytically removed during or just after translocation into the mitochondrial intermembrane space (IMS) by the mitochondrial processing peptidase (MPP) to form the inner-membrane-anchored mature form (AIFmit). During apoptosis, it is further proteolytically processed at amino-acid position 101 leading to the generation of the 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 in a caspase-independent manner.

  • Cellular localization

    Mitochondrion intermembrane space. Mitochondrion inner membrane. Cytoplasm. Nucleus. Cytoplasm > perinuclear region. 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. Colocalizes with EIF3G in the nucleus and perinuclear region.
  • Information by UniProt

  • Database links

    see all

  • Alternative names

    • AIFM1 antibody
    • AIFM1_HUMAN antibody
    • Apoptosis inducing factor 1, mitochondrial antibody
    • Apoptosis inducing factor antibody
    • Apoptosis inducing factor, mitochondrion associated, 1 antibody
    • Apoptosis-inducing factor 1 antibody
    • CMTX4 antibody
    • COWCK antibody
    • COXPD6 antibody
    • Harlequin antibody
    • Hq antibody
    • mAIF antibody
    • MGC111425 antibody
    • MGC5706 antibody
    • mitochondrial antibody
    • Neuropathy, axonal motor-sensory, with deafness and mental retardation antibody
    • neuropathy, axonal, motor-sensory with deafness and mental retardation (Cowchock syndrome) antibody
    • PDCD 8 antibody
    • PDCD8 antibody
    • Programmed cell death 8 (apoptosis inducing factor) antibody
    • Programmed cell death 8 antibody
    • Programmed cell death 8 isoform 1 antibody
    • Programmed cell death 8 isoform 2 antibody
    • Programmed cell death 8 isoform 3 antibody
    • Programmed cell death protein 8 antibody
    • Programmed cell death protein 8 mitochondrial antibody
    • Programmed cell death protein 8 mitochondrial precursor antibody
    • Programmed cell death protein 8 mitochondrial precursor antibody
    • Striatal apoptosis inducing factor antibody
    see all

Images

  • Western blot - Anti-AIF antibody [E20] - Mitochondrial Marker (ab32516)
    Western blot - Anti-AIF antibody [E20] - Mitochondrial Marker (ab32516)
    All lanes : Anti-AIF antibody [E20] - Mitochondrial Marker (ab32516) at 1/1000 dilution

    Lane 1 : Wild-type HEK-293T cell lysate
    Lane 2 : AIFM1 knockout HEK-293T cell lysate

    Lysates/proteins at 20 µg per lane.

    Performed under reducing conditions.

    Predicted band size: 67 kDa
    Observed band size: 67 kDa



    Lanes 1- 2: Merged signal (red and green). Green - ab32516 observed at 67 kDa. Red - Anti-GAPDH antibody [6C5] - Loading Control (ab8245) observed at 37 kDa.

     ab32516 was shown to react with AIF in wild-type HEK-293T cells in western blot. Loss of signal was observed when knockout cell line ab266347 (knockout cell lysate ab256834) was used. Wild-type HEK-293T and AIFM1 knockout HEK-293T cell lysates were subjected to SDS-PAGE. Membrane was blocked for 1 hour at room temperature in 0.1% TBST with 3% non-fat dried milk. ab32516 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) overnight at 4°C at a 1 in 1000 dilution and a 1 in 20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye®800CW) preadsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye®680RD) preadsorbed (ab216776) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.

  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-AIF antibody [E20] - Mitochondrial Marker (ab32516)
    Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-AIF antibody [E20] - Mitochondrial Marker (ab32516)

    Ab32516, at a 1/500 dilution, staining AIF in paraffin embedded human cervical carcinoma tissue by Immunohistochemistry.

    Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.

  • Western blot - Anti-AIF antibody [E20] - Mitochondrial Marker (ab32516)
    Western blot - Anti-AIF antibody [E20] - Mitochondrial Marker (ab32516)
    Anti-AIF antibody [E20] - Mitochondrial Marker (ab32516) at 1/1000 dilution + K562 cell lysate

    Predicted band size: 67 kDa
    Observed band size: 67 kDa

  • Immunoprecipitation - Anti-AIF antibody [E20] - Mitochondrial Marker (ab32516)
    Immunoprecipitation - Anti-AIF antibody [E20] - Mitochondrial Marker (ab32516)
    AIF was immunoprecipitated using 0.5mg K562 whole cell extract, 5µg of Rabbit monoclonal to AIF and 50µl of protein G magnetic beads (+). No antibody was added to the control (-).
    The antibody was incubated under agitation with Protein G beads for 10min, K562 whole cell extract lysate diluted in RIPA buffer was added to each sample and incubated for a further 10min under agitation.
    Proteins were eluted by addition of 40µl SDS loading buffer and incubated for 10min at 70oC; 10µl of each sample was separated on a SDS PAGE gel, transferred to a nitrocellulose membrane, blocked with 5% BSA and probed with ab32516.
    Secondary: Mouse monoclonal [SB62a] Secondary Antibody to Rabbit IgG light chain (HRP) (ab99697).
    Band: 67kDa: AIF
  • Immunocytochemistry/ Immunofluorescence - Anti-AIF antibody [E20] - Mitochondrial Marker (ab32516)
    Immunocytochemistry/ Immunofluorescence - Anti-AIF antibody [E20] - Mitochondrial Marker (ab32516)
    ICC/IF image of ab32516 stained HeLa cells. The cells were 4% PFA fixed (10 min) and then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1h to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab32516, 1µg/ml) overnight at +4°C. The secondary antibody (green) was Alexa Fluor® 488 goat anti-rabbit IgG (H+L) used at a 1/1000 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (red) at a 1/200 dilution for 1h. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43µM.

  • Flow Cytometry (Intracellular) - Anti-AIF antibody [E20] - Mitochondrial Marker (ab32516)
    Flow Cytometry (Intracellular) - Anti-AIF antibody [E20] - Mitochondrial Marker (ab32516)

    Overlay histogram showing K562 cells stained with ab32516 (red line). The cells were fixed with 4% paraformaldehyde (10 min) and then permeabilized with 0.1% PBS-Tween for 20 min. The cells were then incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions followed by the antibody (ab32516, 1/50 dilution) for 30 min at 22°C. The secondary antibody used was DyLight® 488 goat anti-rabbit IgG (H+L) (ab96899) at 1/500 dilution for 30 min at 22°C. Isotype control antibody (black line) was rabbit monoclonal IgG (1µg/1x106 cells) used under the same conditions. Acquisition of >5,000 events was performed. This antibody gave a positive signal in K562 cells fixed with methanol (5 min)/permeabilized with 0.1% PBS-Tween used under the same conditions.

     

  • Anti-AIF antibody [E20] - Mitochondrial Marker (ab32516)
    Anti-AIF antibody [E20] - Mitochondrial Marker (ab32516)

Protocols

To our knowledge, customised protocols are not required for this product. Please try the standard protocols listed below and let us know how you get on.

Click here to view the general protocols

References (59)

Publishing research using ab32516? Please let us know so that we can cite the reference in this datasheet.

ab32516 has been referenced in 59 publications.

  • Xing Y  et al. PAK5-mediated AIF phosphorylation inhibits its nuclear translocation and promotes breast cancer tumorigenesis. Int J Biol Sci 17:1315-1327 (2021). PubMed: 33867848
  • Li Y  et al. microRNA-130a-5p suppresses myocardial ischemia reperfusion injury by downregulating the HMGB2/NF-?B axis. BMC Cardiovasc Disord 21:121 (2021). PubMed: 33658008
  • Zhou H  et al. Upconversion NIR-II fluorophores for mitochondria-targeted cancer imaging and photothermal therapy. Nat Commun 11:6183 (2020). PubMed: 33273452
  • Lu X  et al. Modulation of HOXA9 after skeletal muscle denervation and reinnervation. Am J Physiol Cell Physiol 318:C1154-C1165 (2020). PubMed: 32233950
  • Piao M  et al. Sevoflurane Exposure Induces Neuronal Cell Parthanatos Initiated by DNA Damage in the Developing Brain via an Increase of Intracellular Reactive Oxygen Species. Front Cell Neurosci 14:583782 (2020). PubMed: 33424554
View all Publications for this product

Customer reviews and Q&As

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1-8 of 8 Abreviews or Q&A

Western blot abreview for Anti-AIF antibody [E20] - Mitochondrial Marker

 Excellent
Abreviews
abreview image
Application
Western blot
Sample
Mouse Tissue lysate - whole (Brain, Liver, Heart)
Gel Running Conditions
Reduced Denaturing (12%)
Loading amount
50 µg
Specification
Brain, Liver, Heart
Blocking step
Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 23°C
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Abcam user community

Verified customer

Submitted Feb 25 2020

Immunohistochemistry (Frozen sections) abreview for Anti-AIF antibody [E20] - Mitochondrial Marker

 Good
Abreviews
Application
Immunohistochemistry (Frozen sections)
Sample
Mouse Tissue sections (Skeletal muscle)
Permeabilization
Yes - 0.5% triton X-100
Specification
Skeletal muscle
Blocking step
Normal goat serum as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 10% · Temperature: 24°C
Fixative
Paraformaldehyde
Read More

MS. Anna Perez

Verified customer

Submitted Jun 20 2017

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) abreview for Anti-AIF antibody [E20]

 Good
Abreviews
abreview image
Application
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)
Sample
Human Tissue sections (skin)
Specification
skin
Fixative
Paraformaldehyde
Antigen retrieval step
Heat mediated - Buffer/Enzyme Used: 100mM sodium cytrate
Blocking step
BSA as blocking agent for 2 hour(s) and 0 minute(s) · Concentration: 10% · Temperature: RT°C
Read More

Ms. Hadas Pahima

Verified customer

Submitted Oct 11 2012

Western blot abreview for Anti-AIF antibody [E20]

 Excellent
Abreviews
abreview image
Application
Western blot
Sample
Mouse Tissue lysate - whole (brain)
Loading amount
30 µg
Specification
brain
Gel Running Conditions
Reduced Denaturing (10)
Blocking step
Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: RT°C
Read More

Ilan Sela

Verified customer

Submitted Aug 09 2012

Western blot abreview for Anti-AIF antibody [E20]

 Good
Abreviews
abreview image
Application
Western blot
Sample
Rat Tissue lysate - whole (Skeletal Muscle)
Loading amount
30 µg
Specification
Skeletal Muscle
Gel Running Conditions
Reduced Denaturing (10%)
Blocking step
Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 0.5% · Temperature: RT°C
Read More

Abcam user community

Verified customer

Submitted May 15 2009

Question

Hello,
just to let you know that I have tried the AIF antibody (ab32516) and blotted for CHC (ab21679) as well. When I probe the membrane for both antibodies at the same time, I do not get a band (or just a weak band, even when a high amount of protein is present), but when I probe for the proteins individually everything seems to be ok.

I think and hope that this kind of information could be added to your datasheets, so that this antibody-incompatibility does not lead to surprising results.

Best wishes,

Read More
Answer

Thank you very much for contacting us and for your feedback.

We have published this feedback in the scientific support section of the datasheet and it is now available to all the scientific community. Next, we would like to encourage you to publish Abreviews. Once published you can collect Abpoints that can be exchanged with amazon vouchers.

I hope this information will be helpful. We will look forward to receive your abreviews soon.

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Question

I am interested in looking at AIF as a potential marker of caspase-independent apoptosis. Please can you tell me if this is a reliable marker or if there is another product you would recommend? I don’t see any caspase cleavage but I do see mitochondrial membrane depolarisation and membrane blebbing in my experiments suggesting that some other form of death is occurring.

Also, to use it for this purpose would it be best to look for it in mitochondrial and nuclear fractions by immunoblotting as I presume that FACS & standard immunoblotting would only tell you if it was present in the cell or not or do levels increase after mitochondrial release? Would your cellular fractionation kit ab109719 be suitable for looking at AIF in these fractions?

I am working with rat cells, which of your antibodies is best for this cell type: ab119917, ab32516 or ab1998?

Sorry this is not an area that I am familiar with!

Thanks

Read More
Answer

Thank you for contacting us.

I would suggested searching the potential of this as caspase-independent apoptosis marker in publications. There are many publications related to this protein. Please have a look in pubmed.

As the process triggering apoptosis involves the mitochondrial release of AIF through the mitochondrial membrane, which enters the cytosol, and finally ends up in the cell nucleus where it signals the cell to condense its chromosomes and fragment its DNA molecules in order to prepare for cell death. So the idea would be to compare the AIF level in mitochondrial, cytosolic and nuclear preparations. The kit ab109719 should be able to separate the mitochondrial, cytosolic and nuclear fractions for experiment.

Monoclonal antibodies provide clean WB results so monoclonal will be best suitable e.g. ab119917 and ab32516. This however does not mean polyclonal aren't good, they are good but monoclonal are better.

I hope this information is helpful to you. Please do not hesitate to contact us if you need any more advice or information.

Use our products? Submit an Abreview. Earn rewards!
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Question

Leider ist die Chargennummer nicht mehr aufzufinden, beim Aliquotieren wurde das Originalvial leider weggeschmissen. Alles, was ich weiß ist, dass der Antikörper Anfang März 2010 bestellt wurde. Genügt das, um die Konzentration zu ermitteln? mfg

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Answer

Vielen Dank für Ihre Antwort. Ich konnte anhand Ihrer Angaben die wahrscheinlichste Charge ausfindig machen: Die Konzentration Ihrer Charge ist xxxx. Ich möchte Sie darauf hinweisen, dass diese scheinbar niedrige Konzentration völlig ausreichend ist. Dieser Antikörper ist ein monoklonaler Antikörper aus dem Kaninchen. Kaninchen produzieren extrem spezifische Antikörper mit einer sehr starken Affinität und somit wird weniger Antikörper benötigt als mit monoklonalen aus der Maus. Die Menge an "Tests", die mit einem Röhrchen durchgeführt werden kann, wird von uns streng kontrolliert und immer vergleichbar gehalten. Ich hoffe, dies hilft Ihnen weiter. Bitte zögern Sie nicht, sich wieder bei uns zu melden, falls Sie weitere Fragen haben.

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