Immunofluorescence analysis of AIF was done on 70% confluent log phase HeLa cells. The cells were fixed with 4% paraformaldehyde for 15 minutes; permeabilized with 0.25% Triton™ X-100 for 10 minutes followed by blocking with 5% BSA for 1 hour at room temperature. The cells were incubated with AIF Mouse Monoclonal Antibody (456200) at 1 µg - 2 µg in 1% BSA and incubated for 3 hours at room temperature and then labeled with Alexa Flour® 488 Rabbit Anti-Mouse IgG Secondary Antibody (A11059) at a dilution of 1:400 for 30 minutes at room temperature (Panel a: green). Nuclei (Panel b: blue) were stained with SlowFade® Gold Antifade Mountant with DAPI (S36938). F-actin (Panel c: red) was stained with Alexa Fluor® 594 Phalloidin (A12381). Panel d is a merged image showing cytoplasmic localization of AIF. Panel e shows no primary antibody control. The images were captured at 20X magnification.
|Tested species reactivity||Bovine, C. elegans, Human, Mouse, Rat|
|Host / Isotype||Mouse / IgG1|
|Immunogen||Human heart mitochondria|
|Storage buffer||HEPES buffered saline|
|Contains||0.02% sodium azide|
|Storage Conditions||4° C, do not freeze|
|Tested Applications||Dilution *|
|Immunocytochemistry (ICC)||5-10 ug/ml|
|Immunofluorescence (IF)||Assay Dependent|
|Western Blot (WB)||2-5 ug/ml|
* Suggested working dilutions are given as a guide only. It is recommended that the user titrate the product for use in their own experiment using appropriate negative and positive controls.
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.
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