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Protocol |
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This kit is used to quantitate the amount of AIF in a human sample. It uses the well-established sandwich ELISA format, whereby a pair of monoclonal antibodies, each against a different epitope, is used to capture and then detect the amount of protein in a sample.
AIF is immunocaptured within the wells of a pre-coated microplate and the quantity of enzyme is measured by adding a second AIF-specific antibody that is labeled with horseradish peroxidase. This peroxidase changes the substrate from colorless to blue and the rate of color development is proportional to the amount of protein captured in the well (monitored at 600 nm). Alternatively the assay can be terminated, at a user-defined time, by the addition of 1N HCl (not supplied) and the assay performed as an end point measurement at 450 nm.
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Storage |
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Store at 4°C. The kit is stable for 6 months
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Contents/Specifications |
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20X Buffer (Tube 1) 25 mL
10X Blocking Buffer (Tube 2) 8 mL
Development Solution (Tube 3) 20 mL
Detergent 1 mL
20X Detector antibody (Tube A) 1 mL
20X HRP Label (Tube B) 1 mL
96-well pre-coated microplate (12 strips) 1
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Notes |
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Apoptosis-inducing factor, AIF (O95831), is encoded by a single gene on the X chromosome. It is expressed in all tissues and is imported into the mitochondria for localization in the inter-membrane space. AIF is a flavoprotein using FAD as a cofactor with NADH oxidase activity. AIF is essential for efficient bioenergetic metabolism by promoting assembly and maintaining complex I function.
Under certain pro-apoptotic conditions AIF is cleaved by calpains (Ca2+ dependant) and cathepsins (Ca2+ independent). Soluble AIF translocates through the outer membrane into the cytoplasm and in to the nucleus where it participates in chromatin condensation and DNA fragmentation. This programmed cell death is caspase independent since caspase inhibitors fail to block AIF translocation and cell death.
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