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AIF Recombinant Rabbit Monoclonal Antibody [SZ05-01]

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Specification

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Catalog# ET1603-4

AIF Recombinant Rabbit Monoclonal Antibody [SZ05-01]

Application

  • WB

  • IF-Cell

  • IHC-P

  • IP

  • FC

Reactivity

  • Human

  • Mouse

  • Rat

BSA and Azide free
Conjugation

  • unconjugated

This product has been cited in peer reviewed publications, see list HERE

Overview

Product Name

AIF Recombinant Rabbit Monoclonal Antibody [SZ05-01]

Antibody Type

Recombinant Rabbit monoclonal Antibody

Immunogen

Synthetic peptide within Human AIF aa 502-551 / 613.

Species Reactivity

Human, Mouse, Rat

Validated Applications

WB, IF-Cell, IHC-P, IP, FC

Molecular Weight

Predicted band size: 67 kDa

Positive Control

SKOV-3 cell lysate, Daudi cell lysate, MCF-7 cell lysate, Hela cell lysate, C2C12 cell lysate, NIH/3T3 cell lysate, C6 cell lysate, Mouse liver tissue lysate, Rat liver tissue lysate, Mouse kidney tissue lysate, Rat kidney tissue lysate, MCF-7, SKOV-3, C2C12, human liver tissue, human kidney tissue, mouse liver tissue, mouse heart tissue, rat liver tissue, rat kidney tissue, HeLa.

Conjugation

unconjugated

Clone Number

SZ05-01

RRID

AB_3069681

Product Features

Form

Liquid

Concentration

Storage Instructions

Shipped at 4℃. Store at +4℃ short term (1-2 weeks). It is recommended to aliquot into single-use upon delivery. Store at -20℃ long term.

Storage Buffer

1*TBS (pH7.4), 0.05% BSA, 40% Glycerol. Preservative: 0.05% Sodium Azide.

Isotype

IgG

Purification Method

Protein A affinity purified.

Application Dilution

  • WB

  • 1:1,000-1:5,000

  • IF-Cell

  • 1:50-1:200

  • IHC-P

  • 1:1,000

  • FC

  • 1:1,000

  • IP

  • Use at an assay dependent concentration.

Target

Function

A key event in the apoptotic process is the opening of the mitochondrial permeability transition pore, an event that is regulated by Bcl-2 family proteins, resulting in the release of several proteins from the mitochondrial intermembrane space. Several of these proteins participate in apoptosis, including cytochrome c, procaspases 2, 3, and 9, and AIF (apoptosis-inducing factor). AIF has been shown to cause DNA fragmentation and chromatin condensation and to induce the release of cytochrome c and caspase-9 from mitochondria. Bcl-2 overexpression has been shown to prevent the release of AIF from mitochondria, but not to block its apoptogenic activity.

Background References

1. Tang Y et al. Quantitative proteomic analysis of HER2 normal and overexpressing MCF-7 breast cancer cells revealed proteomic changes accompanied with HER2 gene amplification. J Proteomics 91C:200-209 (2013).

2. Kim TW et al. (ADP-ribose) polymerase 1 and AMP-activated protein kinase mediate progressive dopaminergic neuronal degeneration in a mouse model of Parkinson\'s disease. Cell Death Dis 4:e919 (2013).

Sequence Similarity

Belongs to the FAD-dependent oxidoreductase family.

Tissue Specificity

Expressed in all tested tissues. Detected in muscle and skin fibroblasts (at protein level).; [Isoform 3]: Brain specific.; [Isoform 4]: Expressed in all tested tissues except brain.; [Isoform 5]: Isoform 5 is frequently down-regulated in human cancers.

Post-translational Modification

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.; Ubiquitination by XIAP/BIRC4 does not lead to proteasomal degradation. Ubiquitination at Lys-255 by XIAP/BIRC4 blocks its ability to bind DNA and induce chromatin degradation, thereby inhibiting its ability to induce cell death.

Subcellular Location

Mitochondrion intermembrane space, Mitochondrion inner membrane, Cytoplasm, Nucleus, Membrane.

UNIPROT

SwissProt: O95831 Human

SwissProt: Q9Z0X1 Mouse

SwissProt: Q9JM53 Rat

Synonyms

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

Expand

Images

  • Western blot analysis of AIF on different lysates with Rabbit anti-AIF antibody (<a href="/products/ET1603-4" style="font-weight: bold;text-decoration: underline;">ET1603-4</a>) at 1/1,000 dilution.<br /><br />Lane 1: C2C12 cell lysate (20 µg/Lane)<br />Lane 2: NIH/3T3 cell lysate (20 µg/Lane)<br />Lane 3: C6 cell lysate (20 µg/Lane)<br />Lane 4: Mouse liver tissue lysate (40 µg/Lane)<br />Lane 5: Rat liver tissue lysate (40 µg/Lane)<br />Lane 6: Mouse kidney tissue lysate (40 µg/Lane)<br />Lane 7: Rat kidney tissue lysate (40 µg/Lane)<br /><br />Predicted band size: 67 kDa<br />Observed band size: 67/50 kDa<br /><br />Exposure time: 6 seconds; ECL: K1801;<br />4-20% SDS-PAGE gel.<br /><br />Proteins were transferred to a PVDF membrane and blocked with 5% NFDM/TBST for 1 hour at room temperature. The primary antibody (<a href="/products/ET1603-4" style="font-weight: bold;text-decoration: underline;">ET1603-4</a>) at 1/500 dilution was used in 5% NFDM/TBST at 4℃ overnight. Goat Anti-Rabbit IgG - HRP Secondary Antibody (<a href="/products/HA1001" style="font-weight: bold;text-decoration: underline;">HA1001</a>) at 1/50,000 dilution was used for 1 hour at room temperature.

    Western blot analysis of AIF on different lysates with Rabbit anti-AIF antibody (ET1603-4) at 1/1,000 dilution.

    Lane 1: C2C12 cell lysate (20 µg/Lane)
    Lane 2: NIH/3T3 cell lysate (20 µg/Lane)
    Lane 3: C6 cell lysate (20 µg/Lane)
    Lane 4: Mouse liver tissue lysate (40 µg/Lane)
    Lane 5: Rat liver tissue lysate (40 µg/Lane)
    Lane 6: Mouse kidney tissue lysate (40 µg/Lane)
    Lane 7: Rat kidney tissue lysate (40 µg/Lane)

    Predicted band size: 67 kDa
    Observed band size: 67/50 kDa

    Exposure time: 6 seconds; ECL: K1801;
    4-20% SDS-PAGE gel.

    Proteins were transferred to a PVDF membrane and blocked with 5% NFDM/TBST for 1 hour at room temperature. The primary antibody (ET1603-4) at 1/500 dilution was used in 5% NFDM/TBST at 4℃ overnight. Goat Anti-Rabbit IgG - HRP Secondary Antibody (HA1001) at 1/50,000 dilution was used for 1 hour at room temperature.

  • <span style="font-weight: bold;">☑ Knockout (KO)</span><br /><br />All lanes: Western blot analysis of AIF with anti-AIF antibody (<a href="/products/ET1603-4" style="font-weight: bold;text-decoration: underline;">ET1603-4</a>) at 1/500 dilution.<br /><br />Lane 1: Wild-type Hela whole cell lysate.<br />Lane 2: AIF knockout Hela whole cell lysate.<br /><br /><a href="/products/ET1603-4" style="font-weight: bold;text-decoration: underline;">ET1603-4</a> was shown to specifically react with AIF in Wild-type Hela cells. No band was observed when AIF knockout sample was tested. Wild-type and AIF knockout samples were subjected to SDS-PAGE. Proteins were transferred to a PVDF membrane and blocked with 5% NFDM in TBST for 1 hour at room temperature. The primary Anti-AIF antibody (<a href="/products/ET1603-4" style="font-weight: bold;text-decoration: underline;">ET1603-4</a>, 1/500) and Anti-HSP90 antibody (<a href="/products/ET1605-56" style="font-weight: bold;text-decoration: underline;">ET1605-56</a>, 1/10,000) were used in 5% BSA at room temperature for 2 hours. Goat Anti-Rabbit IgG H&L (HRP) Secondary Antibody (<a href="/products/HA1001" style="font-weight: bold;text-decoration: underline;">HA1001</a>) at 1:300,000 dilution was used for 1 hour at room temperature.

    ☑ Knockout (KO)

    All lanes: Western blot analysis of AIF with anti-AIF antibody (ET1603-4) at 1/500 dilution.

    Lane 1: Wild-type Hela whole cell lysate.
    Lane 2: AIF knockout Hela whole cell lysate.

    ET1603-4 was shown to specifically react with AIF in Wild-type Hela cells. No band was observed when AIF knockout sample was tested. Wild-type and AIF knockout samples were subjected to SDS-PAGE. Proteins were transferred to a PVDF membrane and blocked with 5% NFDM in TBST for 1 hour at room temperature. The primary Anti-AIF antibody (ET1603-4, 1/500) and Anti-HSP90 antibody (ET1605-56, 1/10,000) were used in 5% BSA at room temperature for 2 hours. Goat Anti-Rabbit IgG H&L (HRP) Secondary Antibody (HA1001) at 1:300,000 dilution was used for 1 hour at room temperature.

  • Immunocytochemistry analysis of SKOV-3 cells labeling AIF with Rabbit anti-AIF antibody (<a href="/products/ET1603-4" style="font-weight: bold;text-decoration: underline;">ET1603-4</a>) at 1/50 dilution.<br /><br />Cells were fixed in 4% paraformaldehyde for 10 minutes at 37 ℃, permeabilized with 0.05% Triton X-100 in PBS for 20 minutes, and then blocked with 2% negative goat serum for 30 minutes at room temperature. Cells were then incubated with Rabbit anti-AIF antibody (<a href="/products/ET1603-4" style="font-weight: bold;text-decoration: underline;">ET1603-4</a>) at 1/50 dilution in 2% negative goat serum overnight at 4 ℃. Goat Anti-Rabbit IgG H&L (iFluor&trade; 488, <a href="/products/HA1121" style="font-weight: bold;text-decoration: underline;">HA1121</a>) was used as the secondary antibody at 1/1,000 dilution. PBS instead of the primary antibody was used as the secondary antibody only control. Nuclear DNA was labelled in blue with DAPI.

    Immunocytochemistry analysis of SKOV-3 cells labeling AIF with Rabbit anti-AIF antibody (ET1603-4) at 1/50 dilution.

    Cells were fixed in 4% paraformaldehyde for 10 minutes at 37 ℃, permeabilized with 0.05% Triton X-100 in PBS for 20 minutes, and then blocked with 2% negative goat serum for 30 minutes at room temperature. Cells were then incubated with Rabbit anti-AIF antibody (ET1603-4) at 1/50 dilution in 2% negative goat serum overnight at 4 ℃. Goat Anti-Rabbit IgG H&L (iFluor™ 488, HA1121) was used as the secondary antibody at 1/1,000 dilution. PBS instead of the primary antibody was used as the secondary antibody only control. Nuclear DNA was labelled in blue with DAPI.

  • Immunocytochemistry analysis of C2C12 cells labeling AIF with Rabbit anti-AIF antibody (<a href="/products/ET1603-4" style="font-weight: bold;text-decoration: underline;">ET1603-4</a>) at 1/100 dilution.<br /><br />Cells were fixed in 4% paraformaldehyde for 15 minutes at room temperature, permeabilized with 0.1% Triton X-100 in PBS for 15 minutes at room temperature, then blocked with 1% BSA in 10% negative goat serum for 1 hour at room temperature. Cells were then incubated with Rabbit anti-AIF antibody (<a href="/products/ET1603-4" style="font-weight: bold;text-decoration: underline;">ET1603-4</a>) at 1/100 dilution in 1% BSA in PBST overnight at 4 ℃. Goat Anti-Rabbit IgG H&L (iFluor&trade; 488, <a href="/products/HA1121" style="font-weight: bold;text-decoration: underline;">HA1121</a>) was used as the secondary antibody at 1/1,000 dilution. PBS instead of the primary antibody was used as the secondary antibody only control. Nuclear DNA was labelled in blue with DAPI.<br /><br />Beta tubulin (<a href="/products/HA601187" style="font-weight: bold;text-decoration: underline;">HA601187</a>, red) was stained at 1/100 dilution overnight at +4℃. Goat Anti-Mouse IgG H&L (iFluor&trade; 594, <a href="/products/HA1126" style="font-weight: bold;text-decoration: underline;">HA1126</a>) was used as the secondary antibody at 1/1,000 dilution.

    Immunocytochemistry analysis of C2C12 cells labeling AIF with Rabbit anti-AIF antibody (ET1603-4) at 1/100 dilution.

    Cells were fixed in 4% paraformaldehyde for 15 minutes at room temperature, permeabilized with 0.1% Triton X-100 in PBS for 15 minutes at room temperature, then blocked with 1% BSA in 10% negative goat serum for 1 hour at room temperature. Cells were then incubated with Rabbit anti-AIF antibody (ET1603-4) at 1/100 dilution in 1% BSA in PBST overnight at 4 ℃. Goat Anti-Rabbit IgG H&L (iFluor™ 488, HA1121) was used as the secondary antibody at 1/1,000 dilution. PBS instead of the primary antibody was used as the secondary antibody only control. Nuclear DNA was labelled in blue with DAPI.

    Beta tubulin (HA601187, red) was stained at 1/100 dilution overnight at +4℃. Goat Anti-Mouse IgG H&L (iFluor™ 594, HA1126) was used as the secondary antibody at 1/1,000 dilution.

  • Immunohistochemical analysis of paraffin-embedded human liver tissue with Rabbit anti-AIF antibody (<a href="/products/ET1603-4" style="font-weight: bold;text-decoration: underline;">ET1603-4</a>) at 1/1,000 dilution.<br /><br />The section was pre-treated using heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0) for 20 minutes. The tissues were blocked in 1% BSA for 20 minutes at room temperature, washed with ddH<sub>2</sub>O and PBS, and then probed with the primary antibody (<a href="/products/ET1603-4" style="font-weight: bold;text-decoration: underline;">ET1603-4</a>) at 1/1,000 dilution for 1 hour at room temperature. The detection was performed using an HRP conjugated compact polymer system. DAB was used as the chromogen. Tissues were counterstained with hematoxylin and mounted with DPX.

    Immunohistochemical analysis of paraffin-embedded human liver tissue with Rabbit anti-AIF antibody (ET1603-4) at 1/1,000 dilution.

    The section was pre-treated using heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0) for 20 minutes. The tissues were blocked in 1% BSA for 20 minutes at room temperature, washed with ddH2O and PBS, and then probed with the primary antibody (ET1603-4) at 1/1,000 dilution for 1 hour at room temperature. The detection was performed using an HRP conjugated compact polymer system. DAB was used as the chromogen. Tissues were counterstained with hematoxylin and mounted with DPX.

  • Immunohistochemical analysis of paraffin-embedded human kidney tissue with Rabbit anti-AIF antibody (<a href="/products/ET1603-4" style="font-weight: bold;text-decoration: underline;">ET1603-4</a>) at 1/1,000 dilution.<br /><br />The section was pre-treated using heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0) for 20 minutes. The tissues were blocked in 1% BSA for 20 minutes at room temperature, washed with ddH<sub>2</sub>O and PBS, and then probed with the primary antibody (<a href="/products/ET1603-4" style="font-weight: bold;text-decoration: underline;">ET1603-4</a>) at 1/1,000 dilution for 1 hour at room temperature. The detection was performed using an HRP conjugated compact polymer system. DAB was used as the chromogen. Tissues were counterstained with hematoxylin and mounted with DPX.

    Immunohistochemical analysis of paraffin-embedded human kidney tissue with Rabbit anti-AIF antibody (ET1603-4) at 1/1,000 dilution.

    The section was pre-treated using heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0) for 20 minutes. The tissues were blocked in 1% BSA for 20 minutes at room temperature, washed with ddH2O and PBS, and then probed with the primary antibody (ET1603-4) at 1/1,000 dilution for 1 hour at room temperature. The detection was performed using an HRP conjugated compact polymer system. DAB was used as the chromogen. Tissues were counterstained with hematoxylin and mounted with DPX.

  • Immunohistochemical analysis of paraffin-embedded rat liver tissue with Rabbit anti-AIF antibody (<a href="/products/ET1603-4" style="font-weight: bold;text-decoration: underline;">ET1603-4</a>) at 1/1,000 dilution.<br /><br />The section was pre-treated using heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0) for 20 minutes. The tissues were blocked in 1% BSA for 20 minutes at room temperature, washed with ddH<sub>2</sub>O and PBS, and then probed with the primary antibody (<a href="/products/ET1603-4" style="font-weight: bold;text-decoration: underline;">ET1603-4</a>) at 1/1,000 dilution for 1 hour at room temperature. The detection was performed using an HRP conjugated compact polymer system. DAB was used as the chromogen. Tissues were counterstained with hematoxylin and mounted with DPX.

    Immunohistochemical analysis of paraffin-embedded rat liver tissue with Rabbit anti-AIF antibody (ET1603-4) at 1/1,000 dilution.

    The section was pre-treated using heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0) for 20 minutes. The tissues were blocked in 1% BSA for 20 minutes at room temperature, washed with ddH2O and PBS, and then probed with the primary antibody (ET1603-4) at 1/1,000 dilution for 1 hour at room temperature. The detection was performed using an HRP conjugated compact polymer system. DAB was used as the chromogen. Tissues were counterstained with hematoxylin and mounted with DPX.

  • Immunohistochemical analysis of paraffin-embedded rat kidney tissue with Rabbit anti-AIF antibody (<a href="/products/ET1603-4" style="font-weight: bold;text-decoration: underline;">ET1603-4</a>) at 1/1,000 dilution.<br /><br />The section was pre-treated using heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0) for 20 minutes. The tissues were blocked in 1% BSA for 20 minutes at room temperature, washed with ddH<sub>2</sub>O and PBS, and then probed with the primary antibody (<a href="/products/ET1603-4" style="font-weight: bold;text-decoration: underline;">ET1603-4</a>) at 1/1,000 dilution for 1 hour at room temperature. The detection was performed using an HRP conjugated compact polymer system. DAB was used as the chromogen. Tissues were counterstained with hematoxylin and mounted with DPX.

    Immunohistochemical analysis of paraffin-embedded rat kidney tissue with Rabbit anti-AIF antibody (ET1603-4) at 1/1,000 dilution.

    The section was pre-treated using heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0) for 20 minutes. The tissues were blocked in 1% BSA for 20 minutes at room temperature, washed with ddH2O and PBS, and then probed with the primary antibody (ET1603-4) at 1/1,000 dilution for 1 hour at room temperature. The detection was performed using an HRP conjugated compact polymer system. DAB was used as the chromogen. Tissues were counterstained with hematoxylin and mounted with DPX.

  • Flow cytometric analysis of HeLa cells labeling AIF.<br /><br />Cells were fixed and permeabilized. Then stained with the primary antibody (<a href="/products/ET1603-4" style="font-weight: bold;text-decoration: underline;">ET1603-4</a>, 1/1,000) (red) compared with Rabbit IgG Isotype Control (green). After incubation of the primary antibody at +4℃ for an hour, the cells were stained with a iFluor&trade; 488 conjugate-Goat anti-Rabbit IgG Secondary antibody (<a href="/products/HA1121" style="font-weight: bold;text-decoration: underline;">HA1121</a>) at 1/1,000 dilution for 30 minutes at +4℃. Unlabelled sample was used as a control (cells without incubation with primary antibody; black).

    Flow cytometric analysis of HeLa cells labeling AIF.

    Cells were fixed and permeabilized. Then stained with the primary antibody (ET1603-4, 1/1,000) (red) compared with Rabbit IgG Isotype Control (green). After incubation of the primary antibody at +4℃ for an hour, the cells were stained with a iFluor™ 488 conjugate-Goat anti-Rabbit IgG Secondary antibody (HA1121) at 1/1,000 dilution for 30 minutes at +4℃. Unlabelled sample was used as a control (cells without incubation with primary antibody; black).

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Citation

Products with the same target and pathway

metafield image

AIF Recombinant Rabbit Monoclonal Antibody [SZ05-01] - BSA and Azide free

Application: WB,IF-Cell,IHC-P,IP,FC

Reactivity: Human,Mouse,Rat

Conjugate: unconjugated