Recombinant
RabMAb

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

Reviews (4)Q&A (3)References (25)

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: ICC/IF, WB, IHC-P, Flow Cyt, IP, IHC-Frmore details
  • Species reactivity
    Reacts with: Mouse, Rat, Human
  • Immunogen

    Synthetic peptide within Human AIF aa 500-600 (C terminal). The exact sequence is proprietary.
    (Peptide available as ab203574)

  • Positive control
    • WB: K562 cell lysate. IHC-P: Human cervical carcinoma tissue.
  • General notes

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

    This product is a recombinant rabbit monoclonal antibody.

Properties

Applications

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
ICC/IF 1/500.
WB 1/1000. Detects a band of approximately 67 kDa (predicted molecular weight: 67 kDa).
IHC-P Use at an assay dependent concentration.
Flow Cyt 1/50.

ab172730 - Rabbit monoclonal IgG, is suitable for use as an isotype control with this antibody.
IP Use at an assay dependent concentration.
IHC-Fr Use at an assay dependent concentration. PubMed: 23118224

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

References

This product has been referenced in:
  • Lehmer C  et al. A novel CHCHD10 mutation implicates a Mia40-dependent mitochondrial import deficit in ALS. EMBO Mol Med 10:N/A (2018). WB . Read more (PubMed: 29789341) »
  • Pittala S  et al. Targeting Liver Cancer and Associated Pathologies in Mice with a Mitochondrial VDAC1-Based Peptide. Neoplasia 20:594-609 (2018). WB, IHC-P ; Mouse . Read more (PubMed: 29747160) »
See all 25 Publications for this product

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

Customer reviews and Q&As

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1-7 of 7 Abreviews or Q&A

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
Abcam guarantees this product to work in the species/application used in this Abreview.
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

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.

Read More

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.

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Western blot abreview for Anti-AIF antibody [E20]

 Excellent
Abreviews
Abcam guarantees this product to work in the species/application used in this Abreview.
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

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

Read More
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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Western blot abreview for Anti-AIF antibody [E20]

 Good
Abreviews
Abcam guarantees this product to work in the species/application used in this Abreview.
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

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