ET1603-4

AIF Recombinant Rabbit Monoclonal Antibody [SZ05-01]

cat.: ET1603-4

Product Type:	Recombinant Rabbit monoclonal IgG, primary antibodies
Species reactivity:	Human, Mouse, Rat
Applications:	WB, IF-Cell, IHC-P, IP, FC
Clonality:	Monoclonal
Clone number:	SZ05-01

Form:	Liquid
Storage condition:	Store at +4℃ after thawing. Aliquot store at -20℃ or -80℃. Avoid repeated freeze / thaw cycles.
Storage buffer:	1*TBS (pH7.4), 0.05% BSA, 40% Glycerol. Preservative: 0.05% Sodium Azide.
Concentration:	1ug/ul
Purification:	Protein A affinity purified.
Molecular weight:	Predicted band size: 67 kDa
Isotype:	IgG
Immunogen:	Synthetic peptide within Human AIF aa 502-551 / 613.
Positive control:	SKOV-3 cell lysate, Daudi cell lysate, MCF-7 cell lysate, Hela cell lysate, MCF-7, SKOV-3, human liver tissue, human kidney tissue, mouse liver tissue, mouse heart tissue, Hela.
Subcellular location:	Mitochondrion intermembrane space, Mitochondrion inner membrane, Cytoplasm, Nucleus, Membrane.
Recommended Dilutions: WB IF-Cell IHC-P FC IP	1:500-1:5,000 1:50-1:200 1:50-1:1,000 1:50-1:100 Use at an assay dependent concentration.
Uniprot #:	SwissProt: O95831 Human \| Q9Z0X1 Mouse \| Q9JM53 Rat
Alternative names:	AIFM1 AIFM1_HUMAN Apoptosis inducing factor 1, mitochondrial Apoptosis inducing factor Apoptosis inducing factor, mitochondrion associated, 1 Apoptosis-inducing factor 1 CMTX4 COWCK COXPD6 Harlequin Hq mAIF MGC111425 MGC5706 mitochondrial Neuropathy, axonal motor-sensory, with deafness and mental retardation neuropathy, axonal, motor-sensory with deafness and mental retardation (Cowchock syndrome) PDCD 8 PDCD8 Programmed cell death 8 (apoptosis inducing factor) Programmed cell death 8 Programmed cell death 8 isoform 1 Programmed cell death 8 isoform 2 Programmed cell death 8 isoform 3 Programmed cell death protein 8 Programmed cell death protein 8 mitochondrial Programmed cell death protein 8 mitochondrial precursor Programmed cell death protein 8 mitochondrial precursor Striatal apoptosis inducing factor

Images

Fig1: 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.

Fig2: Western blot analysis of AIF on different lysates with Rabbit anti-AIF antibody (ET1603-4) at 1/500 dilution.

Lane 1: SKOV-3 cell lysate
Lane 2: Daudi cell lysate
Lane 3: MCF-7 cell lysate
Lane 4: Hela cell lysate

Lysates/proteins at 10 µg/Lane.

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

Exposure time: 2 minutes;

8% 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 room temperature for 2 hours. Goat Anti-Rabbit IgG - HRP Secondary Antibody (HA1001) at 1:300,000 dilution was used for 1 hour at room temperature.

	Fig3: Immunocytochemistry analysis of MCF-7 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.
	Fig4: 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.
	Fig5: 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.

	Fig6: 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.
	Fig7: Immunohistochemical analysis of paraffin-embedded mouse liver tissue using anti-AIF antibody. The section was pre-treated using heat mediated antigen retrieval with Tris-EDTA buffer (pH 8.0-8.4) for 20 minutes.The tissues were blocked in 5% BSA for 30 minutes at room temperature, washed with ddH₂O and PBS, and then probed with the primary antibody (ET1603-4, 1/50) for 30 minutes 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.
	Fig8: Immunohistochemical analysis of paraffin-embedded mouse heart tissue using anti-AIF antibody. The section was pre-treated using heat mediated antigen retrieval with Tris-EDTA buffer (pH 8.0-8.4) for 20 minutes.The tissues were blocked in 5% BSA for 30 minutes at room temperature, washed with ddH₂O and PBS, and then probed with the primary antibody (ET1603-4, 1/50) for 30 minutes 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.
	Fig9: Flow cytometric analysis of AIF was done on Hela cells. The cells were fixed, permeabilized and stained with the primary antibody (ET1603-4, 1/50) (red). After incubation of the primary antibody at room temperature for an hour, the cells were stained with a Alexa Fluor 488-conjugated Goat anti-Rabbit IgG Secondary antibody at 1/1000 dilution for 30 minutes.Unlabelled sample was used as a control (cells without incubation with primary antibody; black).

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