Caspases,Paracaspases, and Metacaspases Methods and Protocols

8 caspase-8 fused at the C-terminus to YFP. These protocols describe expression and purification from inclusion bodies, followed ...

9 the case for the expression of inactive caspases, the purified protein is >95 % pure following IMAC chromatography (Fig. 2b ...

10 diameter; e.g., Bio-Rad Econo-Pac 0.7 × 5.0- cm), and let the liquid drain. Sequentially rinse the resin with 5 bed volumes o ...

11 of crude inclusion bodies using centrifugation. Proteins are then solubilized using guanidine, and denatured caspase-8 is pur ...

12 series of fractions containing full-length caspase-8-YFP (~84 kDa) and main contaminants eluting prior to the pool of caspase ...

13 Analyze a 10 μL aliquot from each fraction by SDS-PAGE. Pool the purest and most concentrated fractions. Proteins can be kep ...

14 Kinetic parameter determination is a very useful method to com- pare caspase activity based on their primary specificities. T ...

15 is at half its maximum rate. One can also realize that it becomes difficult to saturate an enzyme if KM is relatively high, i ...

16 and techniques are mastered, the full characterization (titration and kinetic parameter determination) can be accomplished in ...

17 (ε 380 nm = 12,600 M−1 cm−1). A 50 μM solution of pure Afc has an absorbance of 0.630 at 380 nm (see Note 19). Correct the i ...

18 because some of them are specific (e.g., restriction enzymes), peptidases are never truly specific, and the substrate used of ...

19 Executioner caspases (caspase-3, caspase-6, and caspase-7) are fully active and dimeric in a buffer that closely mimics the c ...

20 monomers (inactive) from bacteria [ 27 – 30 ], the assay conditions must somehow force the dimerization of the caspase. To re ...

21 Extract the initial rate (straight portion of the fluorescence plotted against time) of every reaction and plot them against ...

22 Many in vitro methods enable the assessment of specificity. However, one must be careful in using the primary specificity of ...

23 Here, a simple protocol is described to characterize the cleavage of a natural substrate by a caspase in vitro. The procedure ...

24 Using a pipettor, gently resuspend the cells and transfer them to a 15 mL conical tube. Recover the cells by centrifugation ...

25 substrate-binding pocket, active site titration requires conditions that force inhibition by Z-VAD-fmk. Caspase-2 can be succ ...

26 insoluble, and the alternative protocol described in Subheading 3.1.2 must be used. Alternatively, F122Y and L123S mutations ...

27 caspases and to understand the impact of specific cleavage events on caspase activity and specificity. Indeed, although it wa ...