ProSci

AIF Antibody

Product Code:
 
PSI-2301
Product Group:
 
Primary Antibodies
Supplier:
 
ProSci
Host Type:
 
Rabbit
Antibody Isotype:
 
IgG
Antibody Clonality:
 
Polyclonal
Regulatory Status:
 
RUO
Applications:
  • Enzyme-Linked Immunosorbent Assay (ELISA)
  • Immunohistochemistry (IHC)
  • Western Blot (WB)
1 / 10
<strong>Figure 1 Western Blot Validation in Human Cell Lines</strong><br> Loading: 15 μg of lysates per lane. Antibodies: AIF 2301, (2 ug/mL), 1h incubation at RT in 5% NFDM/TBST. Secondary: Goat anti-rabbit IgG HRP conjugate at 1:10000 dilution.
2 / 10
<strong>Figure 2 Independent Antibody Validation (IAV) via Protein Expression Profile in Cell Lines</strong><br> Loading: 15 μg of lysates per lane. Antibodies: AIF 2267, (1 μg/mL), AIF 2239, (1 μg/mL), AIF 2301, (2 μg/mL), and beta-actin (1 μg/mL), 1h incubation at RT in 5% NFDM/TBST. Secondary: Goat anti-rabbit IgG HRP conjugate at 1:10000 dilution.
3 / 10
<strong>Figure 3 Western Blot Validation in Mouse and Rat Cell Lines</strong><br> Loading: 15 μg of lysates per lane. Antibodies: AIF 2301, (2 μg/mL), 1h incubation at RT in 5% NFDM/TBST. Secondary: Goat anti-rabbit IgG HRP conjugate at 1:10000 dilution.
4 / 10
<strong>Figure 4 Western Blot Validation in K562 Cell Line</strong><br> Loading: 15 ug of lysates per lane. Antibodies: AIF 2301, (A: 0.5 μg/mL, B: 1 μg/mL, C: 2 μg/mL), 1h incubation at RT in 5% NFDM/TBST. Secondary: Goat anti-rabbit IgG HRP conjugate at 1:10000 dilution.
5 / 10
<strong>Figure 5 Immunocytochemistry Validation of AIF in K562 Cells</strong><br> Immunocytochemical analysis of K562 cells using anti-AIF antibody (2301) at 5 μg/ml. Cells was fixed with formaldehyde and blocked with 10% serum for 1 h at RT; antigen retrieval was by heat mediation with a citrate buffer (pH6). Samples were incubated with primary antibody overnight at 4˚C. A goat anti-rabbit IgG H&L (HRP) at 1/250 was used as secondary. Counter stained with Hematoxylin.
6 / 10
<strong>Figure 6 KD and Induced Validation of AIF in H1299 Cells(Stambolsky et al., 2006) </strong><br> Western blot analysis of AIF knockdown with anti-AIF antibodies in H1299 cells. AIF expression was disrupted in AIF knockdown cells (siRNA1 and siRNA4). An increased expression of AIF was induced by ZnCl2 treatment, which was not observed in AIF knockdown cells.
7 / 10
<strong>Figure 7 KD Validation of AIF in AIF Silenced Stable Cells(Apostolova et al., 2006) </strong><br> AIF silencing is sustained in stable cell lines. Western blot analysis of stable lines AIF-1-10, AIF-2-4 and pU6-2 using anti-AIF antibodies. AIF protein was disrupted after AIF silencing with AIF siRNA (AIF-1-10 and AIF-2-4) as compared to control (Hep3B and pU6-2).
8 / 10
<strong>Figure 8 Immunofluorescence Validation of AIF in Rat Hippocampal Neurons (Hofer et al., 2011)</strong><br> (G-L) After exposure to bacterial components, AIF colocalized in mature neurons (MAP2; I, L), immature neurons (DcX; H, K), and stem/progenitor cells (Nestin; G, J). AIF expression was detected by anti-AIF antibodies.
9 / 10
<strong>Figure 9 Subcellular Localization Validation of AIF in mononuclear cells (Gupta et al., 2003) </strong><br> A shows mononuclear cells (MNCs) alone, B shows MNCs transfected with control plasmid, C shows MNCs transfected with Bcl-2 expression plasmid. Overlay is of Mitotracker (red) and AIF (green). Hoechst 33258 dye is used to examine chromatin fragmentation. The release of AIF form mitochondria is detected by anti-AIF antibodies.
10 / 10
<strong>Figure 10 Induced Expression Validation of AIF in U937 Cells (Ikai et al., 2006) </strong><br> Release of AIF at 48 h after the treatment with 30 uM magnolol examined by Western blot Analysis with anti-AIF antibodies. AIF release was markedly increased 48h after magnolol treatment.
❮❯

<strong>Figure 1 Western Blot Validation in Human Cell Lines</strong><br> Loading: 15 μg of lysates per lane. Antibodies: AIF 2301, (2 ug/mL), 1h incubation at RT in 5% NFDM/TBST. Secondary: Goat anti-rabbit IgG HRP conjugate at 1:10000 dilution.
<strong>Figure 2 Independent Antibody Validation (IAV) via Protein Expression Profile in Cell Lines</strong><br> Loading: 15 μg of lysates per lane. Antibodies: AIF 2267, (1 μg/mL), AIF 2239, (1 μg/mL), AIF 2301, (2 μg/mL), and beta-actin (1 μg/mL), 1h incubation at RT in 5% NFDM/TBST. Secondary: Goat anti-rabbit IgG HRP conjugate at 1:10000 dilution.
<strong>Figure 3 Western Blot Validation in Mouse and Rat Cell Lines</strong><br> Loading: 15 μg of lysates per lane. Antibodies: AIF 2301, (2 μg/mL), 1h incubation at RT in 5% NFDM/TBST. Secondary: Goat anti-rabbit IgG HRP conjugate at 1:10000 dilution.
<strong>Figure 4 Western Blot Validation in K562 Cell Line</strong><br> Loading: 15 ug of lysates per lane. Antibodies: AIF 2301, (A: 0.5 μg/mL, B: 1 μg/mL, C: 2 μg/mL), 1h incubation at RT in 5% NFDM/TBST. Secondary: Goat anti-rabbit IgG HRP conjugate at 1:10000 dilution.
<strong>Figure 5 Immunocytochemistry Validation of AIF in K562 Cells</strong><br> Immunocytochemical analysis of K562 cells using anti-AIF antibody (2301) at 5 μg/ml. Cells was fixed with formaldehyde and blocked with 10% serum for 1 h at RT; antigen retrieval was by heat mediation with a citrate buffer (pH6). Samples were incubated with primary antibody overnight at 4˚C. A goat anti-rabbit IgG H&L (HRP) at 1/250 was used as secondary. Counter stained with Hematoxylin.
<strong>Figure 6 KD and Induced Validation of AIF in H1299 Cells(Stambolsky et al., 2006) </strong><br> Western blot analysis of AIF knockdown with anti-AIF antibodies in H1299 cells. AIF expression was disrupted in AIF knockdown cells (siRNA1 and siRNA4). An increased expression of AIF was induced by ZnCl2 treatment, which was not observed in AIF knockdown cells.
<strong>Figure 7 KD Validation of AIF in AIF Silenced Stable Cells(Apostolova et al., 2006) </strong><br> AIF silencing is sustained in stable cell lines. Western blot analysis of stable lines AIF-1-10, AIF-2-4 and pU6-2 using anti-AIF antibodies. AIF protein was disrupted after AIF silencing with AIF siRNA (AIF-1-10 and AIF-2-4) as compared to control (Hep3B and pU6-2).
<strong>Figure 8 Immunofluorescence Validation of AIF in Rat Hippocampal Neurons (Hofer et al., 2011)</strong><br> (G-L) After exposure to bacterial components, AIF colocalized in mature neurons (MAP2; I, L), immature neurons (DcX; H, K), and stem/progenitor cells (Nestin; G, J). AIF expression was detected by anti-AIF antibodies.
<strong>Figure 9 Subcellular Localization Validation of AIF in mononuclear cells (Gupta et al., 2003) </strong><br> A shows mononuclear cells (MNCs) alone, B shows MNCs transfected with control plasmid, C shows MNCs transfected with Bcl-2 expression plasmid. Overlay is of Mitotracker (red) and AIF (green). Hoechst 33258 dye is used to examine chromatin fragmentation. The release of AIF form mitochondria is detected by anti-AIF antibodies.
<strong>Figure 10 Induced Expression Validation of AIF in U937 Cells (Ikai et al., 2006) </strong><br> Release of AIF at 48 h after the treatment with 30 uM magnolol examined by Western blot Analysis with anti-AIF antibodies. AIF release was markedly increased 48h after magnolol treatment.

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Further Information

Additional Names:
AIF Antibody: Apoptosis-inducing factor 1, Programmed cell death protein 8, AIF, PDCD8
Application Note:
WB: 0.5-2 μg/mL; ICC: 5 μg/mL.

Antibody validated: Western Blot in human, mouse and rat samples; Immunocytochemistry in human samples. All other applications and species not yet tested.
Background:
AIF Antibody: Apoptosis is characterized by several morphological nuclear changes including chromatin condensation and nuclear fragmentation. These changes are triggered by the activation of members of caspase family, caspase activated DNase, and several novel proteins. A novel gene, the product of which causes chromatin condensation and DNA fragmentation, was recently identified, cloned, and designated apoptosis inducing factor (AIF). Like the critical molecules, cytochrome c and caspase-9, in apoptosis, AIF localizes in mitochondria. AIF translocates to the nucleus when apoptosis is induced and induces mitochondria to release the apoptogenic proteins cytochrome c and caspase-9. AIF induces chromatin condensation and DNA fragmentation, which are the hallmarks of apoptosis, of the isolated nucleus and the nucleus in live cells by microinjection. AIF is highly conserved between human and mouse and widely expressed.
Background References:
  • Zamzami et al. Nature 1999; 401:127-8.
  • Susin SA, Lorenzo HK, Zamzami N, et al. Molecular characterization of mitochondrial apoptosis-inducing factor. Nature 1999;397:441-6 (WD0800)
  • Susin et al. Nature 1999; 397:441-6.
  • Daugas et al. FASEB J. 2000; 14:729-39.
Buffer:
AIF Antibody is supplied in PBS containing 0.02% sodium azide.
Concentration:
1 mg/ml
Conjugate:
Unconjugated
DISCLAIMER:
Optimal dilutions/concentrations should be determined by the end user. The information provided is a guideline for product use. This product is for research use only.
Immunogen:
Anti-AIF antibody (2301) was raised against a peptide corresponding to 14 amino acids near the carboxy terminus of human AIF.

The immunogen is located within the last 50 amino acids of AIF.
ISOFORMS:
Human AIF has 6 isoforms, including isoform 1 (613aa, 67kD), isoform 2 (326aa, 36kD), isoform 3 (609aa, 66kD), isoform 4 (324aa, 35kD), isoform 5 (261aa, 28kD), and isoform 6 (237aa, 26kD). Mouse AIF has one isoform (612aa, 67kD) and Rat AIF also has one isoform (612aa, 67kD). 2301 can detect 4 human isoforms (1,2,3,5) and also can detect mouse and rat AIF.
NCBI Gene ID #:
10256
NCBI Official Name:
connector enhancer of kinase suppressor of Ras 1
NCBI Official Symbol:
CNKSR1
NCBI Organism:
Homo sapiens
Physical State:
Liquid
PREDICTED MOLECULAR WEIGHT:
Predicted: 67kD

Observed: 68kD
Protein Accession #:
O95381
Protein GI Number:
50400606
Purification:
AIF Antibody is DEAE purified.
Research Area:
Apoptosis,Cancer
SPECIFICITY:
Multiple isoforms of AIF are known to exist.
Swissprot #:
O95381
User NOte:
Optimal dilutions for each application to be determined by the researcher.
VALIDATION:

Independent Antibody Validation in Cell lines (Figure 2) shows similar AIF expression profile in human and mouse cell lines detected by three independent anti-AIF antibodies that recognize different epitopes, 2267 against central domain, 2239 against N-terminus domain and 2301 against C-terminus domain.  AIF proteins are detected in the most tested cell lines at different expression levels by the three independent antibodies. 

KD Validation (Figure 6,7): Anti-AIF antibody specificity was further verified by AIF specific knockdown. AIF signal in H1299 cells and AIF stable cells transfected with AIF siRNAs was disrupted in comparison with that in cells transfected with control siRNAs.

Localization Validation (Figure 9): AIF detected by anti-AIF antibodies is colocalized with Mitotracker (mitochondrion marker).  AIF is localized in mitochondria.

Induced Expression Validation (Figure 10): AIF expression detected by anit-AIF antibodies was up-regulated by Magnolol treatment.

 

References

  1. Stambolsky et al. Regulation of AIF expression by p53. Cell Death Differ. 2006;13(12):2140-9. Epub 2006. PMID: 16729031
  2. Apostolova et al. Loss of apoptosis-inducing factor leads to an increase in reactive oxygen species, and an impairment of respiration that can be reversed by antioxidants. Cell Death Differ. 2006;13(2):354-7. PMID: 16195738
  3. Hofer et al. Bacterial meningitis impairs hippocampal neurogenesis. J Neuropathol Exp Neurol. 2011;70(10):890-9. doi: 10.1097/NEN.0b013e3182303f31. PMID: 21937913
  4. Gupta et al. Arsenic trioxide induces apoptosis in peripheral blood T lymphocyte subsets by inducing oxidative stress: a role of Bcl-2. Mol Cancer Ther. 2003;2(8):711-9. PMID: 12939460
  5. Ikai et al. Magnolol-induced apoptosis is mediated via the intrinsic pathway with release of AIF from mitochondria in U937 cells. Biol Pharm Bull. 2006;29(12):2498-501. PMID: 17142989
  6. Nebbioso et al. Tumor-selective action of HDAC inhibitors involves TRAIL induction in acute myeloid leukemia cells. Nat Med. 2005;11(1):77-84. Epub 2004. PMID: 15619633
  7. Karlsson et al. Arsenic trioxide-induced death of neuroblastoma cells involves activation of Bax and does not require p53. Clin Cancer Res. 2004;10(9):3179-88. PMID: 15131059
  8. Li et al. Apoptotic signaling pathways induced by nitric oxide in human lymphoblastoid cells expressing wild-type or mutant p53. Cancer Res. 2004;64(9):3022-9. PMID: 15126337
  9. Cervera et al. Cells silenced for SDHB expression display characteristic features of the tumor phenotype. Cancer Res. 2008;68(11):4058-67. doi: 10.1158/0008-5472.CAN-07-5580. PMID: 18519664
  10. Hahn et al. Galectin-1 induces nuclear translocation of endonuclease G in caspase- and cytochrome c-independent T cell death. Cell Death Differ. 2004;11(12):1277-86. PMID: 15297883
  11. Hamacher-Brady et al. Response to myocardial ischemia/reperfusion injury involves Bnip3 and autophagy. Cell Death Differ. 2007;14(1):146-57. Epub 2006. PMID: 16645637
  12. Nakagawa et al. Characterized mechanism of alpha-mangostin-induced cell death: caspase-independent apoptosis with release of endonuclease-G from mitochondria and increased miR-143 expression in human colorectal cancer DLD-1 cells. Bioorg Med Chem. 2007;15(16):5620-8. Epub 2007. PMID: 17553685
  13. Itoh T et al. Eupalinin A isolated from Eupatorium chinense L. induces autophagocytosis in human leukemia HL60 cells. Bioorg Med Chem. 2008;16(2):721-31. Epub 2007. PMID: 17980607
  14. Yel et al. Thimerosal induces neuronal cell apoptosis by causing cytochrome c and apoptosis-inducing factor release from mitochondria. Int J Mol Med. 2005;16(6):971-7. PMID: 16273274
  15. Nakagawa et al. A potent apoptosis-inducing activity of a sesquiterpene lactone, arucanolide, in HL60 cells: a crucial role of apoptosis-inducing factor. J Pharmacol Sci. 2005;97(2):242-52. Epub 2005. PMID: 15699578