Caspases,Paracaspases, and Metacaspases Methods and Protocols

28 4 Notes Black plates are designed to reduce well-to-well cross talk and background for fluorescence assays, whereas white pl ...

29 and caspase-7 to self-process fully as compared to caspase-3. Additionally, better yields are obtained using a slower expres- ...

30 expression time for various caspases. Be consistent with the expression time. A longer or a shorter expression period can inf ...

31 small subunit (~10–12 kDa). The generation of those subunits is the result of the auto-catalytic activation of caspases durin ...

32 LED-based systems are more stable and should be calibrated every 6 months, whereas lamp-based instruments should be calibrat ...

33 used for linear regression result in a good correlation factor (r^2 > 0.98). Most caspase preparations will have a titer t ...

34 required to completely inhibit caspase-3. For example, if the titration was performed using 100 nM caspase-3, an intercept at ...

35 If KM is too high (>200 μM), it is better to determine the kcat/KM value using pseudo-first order conditions. If [S] ≪KM, ...

36 are too diluted for analysis, set up larger sample volumes and precipitate proteins with 10 % trichloroacetic acid (TCA) for ...

37 In E. coli, a fraction of the protein subunits will reassemble into a fully cleaved dimer. This approach can be particularly ...

38 Agard NJ, Wells JA (2009) Methods for the proteomic identification of protease substrates. Curr Opin Chem Biol 13:503–509 De ...

39 Stennicke HR, Salvesen GS (1999) Catalytic properties of the caspases. Cell Death Differ 6:1054–1059 Pop C, Fitzgerald P, Gr ...

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41 Peter V. Bozhkov and Guy Salvesen (eds.), Caspases, Paracaspases, and Metacaspases: Methods and Protocols, Methods in Molecul ...

42 P1′ position. In intact substrates, fl uorescence emission is either absent or very weak. Following protease hydrolysis, fl u ...

43 was followed by the identifi cation of the exact composition of isokinetic mixture determining equimolar levels of substituti ...

44 conducted by Wachmann et al. [ 15 ] with a use of PS-SCL confi rmed this fi nding and established LEHD as the best substrate ...

45 Table 1 Complete tetrapeptide-ACC libraries for studying P4-P2 substrate specifi city of caspases Library Structure Number of ...

46 Rink amide RA resin, particle size 200–300 mesh, loading 0.48 mmol/g ( see Note 7 ). 7-Fmoc-aminocoumarin-4-acetic acid, pur ...

47 3 Methods To ensure that the caspase of interest will display high enough activity during kinetic assay, each of caspase act ...