Caspases,Paracaspases, and Metacaspases Methods and Protocols

48 the assay (its RFU/s will be in the range 0.5–1.0). Substrates displaying RFU/s lower than 1 % of that of the best caspase su ...

49 After 10 min of incubation, transfer the enzyme sample from the 15 ml Falcon tube to the reagent reservoir and add 99 μl in ...

50 Fig. 4 The general scheme for how the linear part of the plot from library screen ing should be selected Marcin Poręba et al. ...

51 Fig. 5 The pattern of P4-P2 SCL spotting on the 96-well plate. This pattern is an alternative to the one presented in Fig. 3 ...

52 Remove N -Fmoc groups by incubating the resin in 5 ml of 20 % piperidine in DMF for 5, 5, and 30 min. Each time, agi- tate g ...

53 Add 3 ml of 20 % piperidine in DMF to synthesis vessel and gently agitate for 5 min. Remove piperidine solution and then rep ...

54 Transfer a few resin beads to a test tube and perform ninhydrin test. If test is positive (beads are blue), repeat steps 9 ...

55 and mix it several times. Repeat this procedure until the last well is fi lled with diluted substrate. Mix solution in H1 wel ...

56 If K (^) M experiment has been carried out at least three times, it is possible to calculate three main substrate kinetic p ...

57 Combinatorial libraries and individual substrates should be brought from −80°C to room temperature very slowly (over at leas ...

58 Silane derivative (TIPS) shows good effi cacy in quenching carbocations. Cleavage should be completed in 2–3 h at room temp ...

59 P1 arginine aminocoumarin substrate library for trypsin-like serine proteases. Bioorg Med Chem Lett 10(20):2291–2294 Backes ...

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

62 Platform [ 1 , 2 ]. In this platform, up-front fractionation of pro- teomic samples is performed by one-dimensional SDS-PAGE ...

63 precise sites-of-cleavage) enables prediction of the functional consequences of cleavage events. Here we describe a detailed ...

64 SEQUEST software. DTASelect software. Custom Perl scripts: coverage.pl, protomap.pl, and peptogra- pher.pl (available at htt ...

65 Add enough 55 mM iodoacetamide (made fresh) to cover gel band (approx 200 μL). Place tubes in the dark for 30 min at room te ...

66 buffer A with a fl ow rate of 0.1 mL/min with a tee splitter to reduce the fl ow rate to 300–400 nL/min. Resuspend peptide s ...

67 Install a Windows-compatible distribution of Perl. The ActivePerl distribution from ActiveState is free of charge and is ava ...