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Also for DIABLO (NM_019887)
|This whole rabbit serum was prepared by repeated immunizations with recombinant His6-tagged human Smac/DIABLO protein (amino acids 56-239).|
|Human HeLa and LNCaP cells
||ELISA: 1:5,000 - 1:20,000, WB: 1:1,000 - 1:2,000, IP: 1:100
|0.02 M Potassium Phosphate, 0.15 M Sodium Chloride, pH 7.2|
|Apoptosis is a conserved cell suicide program essential for the development and homeostasis of multi-cellular organisms. Abnormal inhibition of apoptosis is a hallmark of cancer and autoimmune diseases, whereas excessive cell death is found in neurodegenerative disorders such as Alzheimers disease. Executioners of the apoptotic program are cysteine proteases termed caspases that exist as inactive zymogens in living cells and are activated during apoptosis. Active caspases cleave key intracellular protein substrates, resulting in the characteristic morphological changes associated with apoptosis. The release of cytochrome c from the mitochondria triggers the oligomerization of Apaf-1 in an ATP/dATP-dependent manner and induces the autoactivation of caspase-9. Active caspase-9 in turn activates downstream effector caspases including caspase -3, -6 and -7.
|Homo sapiens diablo, IAP-binding mitochondrial protein (DIABLO), transcript variant 1|
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Western blot using anti-Smac detects a 26 kDa band when 1 µg of recombinant Smac is applied (lane 1). Lane 2 shows Smac detection when 30 µg of 1% NP-40 treated cell lysate from HeLa cells is applied. Lanes 3 & 4 show 30 µg each of cytosolic fractions from HeLa cell lysates both with (lane 3) and without (lane 4) treatment with 30 µM etoposide. Recombinant Smac migrates slower than the native form because of the His6-tag. The blot was incubated overnight with a 1:1000 dilution of anti-Smac in TBST. Detection occurs using a 1:1000 dilution of HRP Goat-a-Rabbit with visualization via ECL. Film exposure approximately 1’
Anti-Smac is shown to detect a 25-26 kDa band in partially purified recombinant human Smac protein by western blot. Lanes 1-3 are loaded with 1, 10 and 100 ng of protein per lane, respectively. The blot was incubated overnight with a 1:1000 dilution of anti-Smac in TBST. Detection occurs using a 1:1000 dilution of HRP Goat-a-Rabbit with visualization via ECL. Film exposure approximately 1’. Other detection systems will yield similar results.