Mitochondria and cytosolic fractions were isolated as described (Cover et al., 2005 (link)). Briefly, the liver was homogenized in ice cold isolation buffer (pH7.4) containing 220 mM mannitol, 70 mM sucrose, 2.5 mM HEPES, 10 mM EDTA, 1 mM EGTA, and 0.1% bovine serum albumin. Mitochondria were isolated by differential centrifugation (20,000 xg) and washed with 2 ml of isolation buffer. The 20,000 xg supernatant was used to evaluate the release of mitochondrial factors such as apoptosis-inducing factor (AIF) by western blotting. Western blotting was carried out as described in detail (Bajt et al., 2000 (link)) using the following antibodies: a rabbit anti-AIF antibody (Abcam, Cambridge, MA), a rabbit anti-Cyp2E1 polyclonal antibody (Abcam, Cambridge, MA), a mouse anti-Bcl-xl monoclonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA), a rabbit anti-LC-3 polyclonal antibody (MBL, Naka-ku Nagoya, Japan), a mouse anti-GADD 153/CHOP monoclonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA), a rat anti-Grp 78 monoclonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA) and a mouse monoclonal anti-metallothionein antibody (DAKO Corp., Carpinteria, CA). A horseradish peroxidase-coupled anti-rabbit IgG (Santa Cruz) was used as secondary antibody. Proteins were visualized by enhanced chemiluminescence (Amersham Pharmacia Biotech. Inc., Piscataway, NJ).
Rabbit anti aif antibody
Manufactured by Abcam
Rabbit anti-AIF antibody is a primary antibody that specifically binds to the Apoptosis-Inducing Factor (AIF) protein. AIF is a mitochondrial flavoprotein that plays a role in programmed cell death. This antibody can be used in various immunoassays to detect and study the AIF protein.
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2 protocols using rabbit anti aif antibody
1
Isolation and Analysis of Mitochondrial Factors
2
Immunofluorescence Assay for AEBP1 Knockdown
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Cells were grown on coverslips and subjected to AEBP1 down regulation as described above. After desired period of incubation, cells were fixed with 4% paraformaldehyde for 20 minutes, permeabilized with 0.1% triton X-100 for 10 minutes and blocked with 1% BSA. Cells were incubated with primary antibodies in 1X PBS with 1% BSA at 4 °C for overnight. The primary antibodies used were rabbit anti-AIF antibody (Abcam), rabbit anti- PI3 Kinase p110 beta antibody (Abcam) and mouse monoclonal anti-γH2AX antibody (raised in house). Appropriate secondary antibodies conjugated with Alexa fluor 488, 568, or 633 (Invitrogen/Molecular Probes) were used. The secondary antibody was added and incubated for one hour at room temperature. Mitotracker Red CMXRos dye (Life Technologies) was used to assess the mitochondrial outer membrane potential (MOMP).
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