Overview

  • Product name
  • Description
    Rabbit polyclonal to AIF
  • Host species
    Rabbit
  • Tested applications
    Suitable for: WB, IHC-P, ICC/IFmore details
  • Species reactivity
    Reacts with: Mouse, Rat, Human
  • Immunogen

    Synthetic peptide corresponding to Human AIF aa 517-531.
    Database link: O95831
    (Peptide available as ab7870)

  • Positive control
    • K562 cell lysate
  • General notes
    Apoptosis Inducing Factor

Properties

Applications

Our Abpromise guarantee covers the use of ab1998 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
WB Use a concentration of 0.25 - 1 µg/ml. Detects a band of approximately 67 kDa (predicted molecular weight: 67 kDa).Can be blocked with AIF peptide (ab7870). Can be blocked with AIF (internal) peptide (human).
IHC-P Use at an assay dependent concentration.
ICC/IF Use a concentration of 1 µg/ml.

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
  • 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

  • All lanes : Anti-AIF antibody (ab1998) at 1 µg/ml

    Lane 1 : K562 cell lysate
    Lane 2 : Rat heart tissue lysate
    Lane 3 : Mouse heart tissue lysate

    Predicted band size: 67 kDa

  • Immunohistochemistry of AIF in human retina with anti-AIF (IN) at 10 µg/ml.
  • ab1998 staining AIF in human U2OS cells by Immunocytochemistry/ Immunofluorescence.
    Samples were fixed using 4% paraformaldehyde.

    Left image shows fixed cells labeled with ab1998 (red) and cyclophilin A (green).
    Right image shows U2OS cell 6 hours after induction of apoptosis by 200 nM staurosporine.
    Note translocated of AIF to the nucleus upon induction of apoptosis.
  • ICC/IF image of ab1998 stained HeLa cells. The cells were 4% formaldehyde 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 (ab1998, 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.

References

This product has been referenced in:
  • Chen S  et al. Alpha lipoic acid attenuates cadmium-induced nephrotoxicity via the mitochondrial apoptotic pathways in rat. J Inorg Biochem 184:19-26 (2018). Read more (PubMed: 29654931) »
  • Soriano J  et al. Cell Death Mechanisms in Tumoral and Non-Tumoral Human Cell Lines Triggered by Photodynamic Treatments: Apoptosis, Necrosis and Parthanatos. Sci Rep 7:41340 (2017). Read more (PubMed: 28112275) »
See all 24 Publications for this product

Customer reviews and Q&As

1-7 of 7 Abreviews or Q&A

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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Answer



ab1998 : https://www.abcam.com/ab1998, where the immunogen is described asa peptide corresponding to amino acids 517 to 531 of human AIF. This sequence is identical tothose of mouse and rat AIF.

ab32042 : https://www.abcam.com/ab32042, where the immunogen is described as : a synthetic peptide corresponding to residues following Ser29 of human Caspase 3 (N terminus of p17 subunit).

ab59348 : https://www.abcam.com/ab59348, where the immunogen is described as a synthesized non phosphopeptide derived from human Bcl2 around the phosphorylation site of threonine 69 (ARTPSP).

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Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)
Sample
Human Tissue sections (Breast cancer tissue and adjacent normal)
Specification
Breast cancer tissue and adjacent normal
Fixative
Formaldehyde
Antigen retrieval step
Other
Permeabilization
No
Blocking step
H2o2 of abcam secondary antibody Kit Expose Kit as blocking agent for 10 minute(s) · Concentration: 1% · Temperature: 37°C

Mrs. Thushara Jayan Pillai

Verified customer

Submitted Oct 24 2011

Abreviews
Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Western blot
Sample
Human Cell lysate - whole cell (SW1573 lung cancer cell line)
Loading amount
50 µg
Specification
SW1573 lung cancer cell line
Gel Running Conditions
Reduced Denaturing (10% SDS-PAGE)
Blocking step
Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 8% · Temperature: 25°C

Abcam user community

Verified customer

Submitted May 27 2011

Answer

Thank you for your email. In your prior message with the attachment containing the Western blot image, incubation for 1 hr at RT was mentioned but not overnight at 4C. I agree with you and I would expect that you would see some sort of signal with your samples and overnight incubation. As we guarantee our products to work as stated on the online datasheet, I would be happy to offer you a free of charge replacement vial or a credit note or refund. Please let me know how you would like to proceed, and happy holidays!

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Question

BATCH NUMBER 108063 ORDER NUMBER 112828 DESCRIPTION OF THE PROBLEM No signal (No band at all) SAMPLE Purified Rat liver Mitochondria PRIMARY ANTIBODY AB 1998 DETECTION METHOD Super signal west pico (pierce) POSITIVE AND NEGATIVE CONTROLS USED We have tried just rat liver mitochondria lysates using triton x 100, SDS or NP 40. This is my model, and the release of cytochrome c works fine, so I am not interested in other species. ANTIBODY STORAGE CONDITIONS The antibody was Aliquoted upon arrival and store at -30 ?C SAMPLE PREPARATION lysis buffer containing PMSF 1 mM, EDTA 10 mM, Nacl 300 mM, BME 5 mM, NP40 1 %,Tris 50 mM (pH: 7.6) plus protease inhibitors cocktail. AMOUNT OF PROTEIN LOADED 5-30 ?g per lane ELECTROPHORESIS/GEL CONDITIONS Std 12 %SDS-PAGE, run at 170 volts during 1 hour. TRANSFER AND BLOCKING CONDITIONS Wet transfer, 25 volts over night. Blocking solution: 5 % Non fat milk in TBS. 1 Hour RT. SECONDARY ANTIBODY Goat anti-Rabbit- HRP conjugated (AP 132P, Chemicon) HOW MANY TIMES HAVE YOU TRIED THE APPLICATION? 4 HAVE YOU RUN A "NO PRIMARY" CONTROL? No WHAT STEPS HAVE YOU ALTERED? NONE ADDITIONAL NOTES I have experience with several anti-bodies. For most of them the western blot condition are the same with not troubles of any kind. I am investigating the release of apoptotic factors from isolated mitochondria induced by Bax. The detection in this experiments of cytochrome c works fine. But now I wanted to try AIF release in this model. But the AB 1998 anti-body did not work.

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Answer

Thank you for your enquiry and I'm sorry to hear that you are experiencing difficulty with ab1998. At this time I would like to make the following suggestions in order to help improve your results. I would suggest extending the incubation period with the primary antibody to overnight at 4C and also increasing the concentration of ab1998 to 1:500. I also suggest running a positive control - K562 cell lysate is recommended for ab1998. Please contact us again if you have any additional questions, and happy holidays.

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Answer

This antibody has not been tested for IHC, therefore we cannot guarantee results.

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