细胞凋亡分析实验指南
Apoptosis Assays
Preparation of the Samples for Cytochrome c ELISA
Materials
Quantikine® Rat/Mouse Cytochrome c ELISA Kit (R&D Systems Catalog # MCTC0)
5% Triton® X-100
Methods
Thaw the samples immediately prior to running the ELISA.
Add 7.5 µL of 5% Triton® X-100 to the total Cytochrome c control sample (75 µL) that was frozen immediately after incubation at 30° C.
Add 5.0 µL of 5% Triton X-100 to the remaining 50 µL samples and controls. Vortex the samples.
Recombinant Mouse Bcl-x
Materials
Recombinant mouse Bcl-xL (R&D Systems Catalog # 878-BC)
Caspase-8-cleaved recombinant mouse BID (R&D Systems Catalog # 883-M8)
Crude or enriched mouse liver mitochondria (See Preparation of Mitochondria from Mouse Liver)
Dilution Buffer: 25 mM HEPES-KOH (pH 7.4), 0.1 M KCl, 10% Glycerol, 1 mg/mL fatty acid free BSA* (Sigma Catalog # A6003)
Recombinant Human BID Cytochrome c Release Assay
Materials
Recombinant human BID (R&D Systems Catalog # 846-BD)
Crude or enriched mouse liver mitochondria (See Preparation of Mitochondria from Mouse Liver)
Dilution Buffer: 25 mM HEPES-KOH (pH 7.4), 0.1 M KCl, 1 mg/mL fatty acid free BSA* (Sigma Catalog # A6003)
Recombinant Human Bcl-x
Materials
Recombinant human Bcl-xL (R&D Systems Catalog # 894-BX)
Caspase-8-cleaved recombinant human BID (R&D Systems Catalog # 882-B8)
Crude or enriched mouse liver mitochondria (See Preparation of Mitochondria from Mouse Liver)、
Dilution Buffer: 25 mM HEPES-KOH (pH 7.4), 0.1 M KCl, 10% Glycerol, 1 mg/mL fatty acid free BSA* (Sigma Catalog # A6003)
Caspase Activity Assays for Inhibitors of Apoptosis & Mitochondrial Proteins
As their name implies, a primary role for members of the Inhibitors of Apoptosis (IAP) family lies in their ability to suppress apoptosis. This occurs through the inhibition of pro-apoptotic members of the Caspase family. IAP proteins are characterized structurally by the presence of at least one BIR (baculoviral IAP repeat) domain. Binding of the BIR domain of IAPs to active caspases inhibits caspase activity. Eight IAPs have been identified in humans including Apollon/BRUCE, cIAP-1, cIAP-2, ILP-2, Livin, NAIP, Survivin, and XIAP.
Recombinant Human Bcl-xL Cytochrome c Release
Materials
Recombinant human Bcl-xL (R&D Systems Catalog # 894-BX)
Caspase-8-cleaved recombinant human BID (R&D Systems Catalog # 882-B8)
Crude or enriched mouse liver mitochondria (See Preparation of Mitochondria from Mouse Liver)
Dilution Buffer: 25 mM HEPES-KOH (pH 7.4), 0.1 M KCl, 10% Glycerol, 1 mg/mL fatty acid free BSA* (Sigma Catalog # A6003)