Caspases,Paracaspases, and Metacaspases Methods and Protocols

68 Create a protomap.params fi le. An example of a protomap.params fi le is shown in Fig. 3. The protomap.params fi le tells th ...

69 (d) Type peptographer.pl alldata.pmap and press <enter> (or use the fi lename you renamed alldata.pmap to). It may take ...

70 4 Notes When preparing cell lysates, add protease and specifi c caspase inhibitors to eliminate any residual proteolytic act ...

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

72 on human caspase-2 indicate that it forms a p19/p12 homodimer in solution with two active sites [ 14 ]. Interestingly the cas ...

73 These studies indicate that the role of caspase-2 in developmental cell death is redundant or can be compensated by other cas ...

74 Caspase-2 processing in apoptotic cells can be detected in apoptotic cell lysates by immunoblotting with specifi c caspase-2 ...

75 Biosciences) supplemented with 10 mM 4-(2-hydroxyethyl)- 1-piperazineethanesulfonic acid (HEPES), 100 mM penicillin/ streptom ...

76 Centrifuge. PBS. Chromatography columns (such as Bio-Rad Poly-prep chro- matography columns). Ice. Sonicator. Glutathione Se ...

77 In vitro-coupled transcription/translation kit, such as the TNT™ Coupled Reticulocyte Lysate System (commercially available ...

78 Power pack supply with constant current optional settings. Wash buffer: PBS with 0.05 % Tween-20 (PBS-T). Membrane blocking ...

79 cells are plated onto LB/agar containing the appropriate antibiotic (ampicillin, kanamycin, and/or chloramphenicol) for selec ...

80 should be used as controls. If assessing caspase-2 activity in protein extracts, controls should also include untreated cells ...

81 coding regions containing the putative caspase-2 cleavage site can be used [ 43 ]. In vitro transcription/translation of eac ...

82 Pellet cells by centrifugation at 200 × g for 5 min and wash in cold PBS. Resuspend cell pellet in cell extraction buffer (a ...

83 pan- caspase inhibitor which will interact with all active caspases and has been used to effi ciently detect active caspase-2 ...

84 recombinant caspases. We have found that the rat anti-caspase-2 antibody clone (11B4) works best to detect full-length cas- p ...

85 There are also several commercial suppliers of purifi ed recombinant caspase-2, such as Enzo Life Sciences (USA), Millipore ...

86 Kim IR, Murakami K, Chen NJ et al (2009) DNA damage- and stress-induced apoptosis occurs independently of PIDD. Apoptosis 14 ...

87 specifi cities of members of the caspase family and granzyme B. Functional relationships established for key mediators of apo ...