Anti-AIF antibody [E20] - Mitochondrial Marker

• IHC-P

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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.

Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - 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.

• ICC/IF

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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.

• IP

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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 70^oC; 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

All lanes:

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

Predicted band size: 67 kDa

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• WB

Supplier Data

Western blot - Anti-AIF antibody [E20] - Mitochondrial Marker (AB32516)

In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control (ab181602) staining at 1/200000 dilution.

In Western blot, Anti-AIF antibody [E20] (ab32516) staining at 1/1000 dilution.

All lanes:

Western blot - Anti-CISD1/MitoNEET antibody [EPR29116-27] (<a href='/en-us/products/primary-antibodies/cisd1-mitoneet-antibody-epr29116-27-ab318199'>ab318199</a>) at 1/1000 dilution

Lane 1:

HeLa (human cervical adenocarcinoma epithelial cell) cytoplasmic fraction at 20 µg

Lane 2:

HeLa mitochondrial fraction at 20 µg

Secondary

All lanes:

Goat Anti-Rabbit IgG (HRP) with minimal cross-reactivity with human IgG at 1/2000 dilution

Observed band size: 15 kDa,36 kDa,67 kDa

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Exposure time: 10s

• WB

Lab

Western blot - Anti-AIF antibody [E20] - Mitochondrial Marker (AB32516)

Lanes 1- 2 : Merged signal (red and green). Green - ab32516 observed at 67 kDa. Red - Anti-GAPDH antibody [6C5] - Loading Control (ab8245) observed at 37 kDa.

ab32516 was shown to react with AIF in wild-type HEK-293T cells in western blot. Loss of signal was observed when knockout cell line ab266347 (knockout cell lysate ab256834) was used. Wild-type HEK-293T and AIFM1 knockout HEK-293T cell lysates were subjected to SDS-PAGE. Membrane was blocked for 1 hour at room temperature in 0.1% TBST with 3% non-fat dried milk. ab32516 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) overnight at 4°C at a 1 in 1000 dilution and a 1 in 20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^®800CW) preadsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^®680RD) preadsorbed (ab216776) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.

All lanes:

Western blot - Anti-AIF antibody [E20] - Mitochondrial Marker (ab32516) at 1/1000 dilution

Lane 1:

Wild-type HEK-293T cell lysate at 20 µg

Lane 2:

AIFM1 knockout HEK-293T cell lysate at 20 µg

Lane 2:

Western blot - Human AIFM1 (AIF) knockout HEK-293T cell line (<a href='/en-us/products/cell-lines/human-aifm1-aif-knockout-hek-293t-cell-line-ab266347'>ab266347</a>)

Predicted band size: 67 kDa

Observed band size: 67 kDa

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• WB

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Western blot - Anti-AIF antibody [E20] - Mitochondrial Marker (AB32516)

All lanes:

Western blot - Anti-AIF antibody [E20] - Mitochondrial Marker (ab32516) at 1/1000 dilution

All lanes:

K562 cell lysate

Predicted band size: 67 kDa

Observed band size: 67 kDa

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