Caspases,Paracaspases, and Metacaspases Methods and Protocols

128 protocol utilizes a pro-luminescent caspase-3/7 substrate (Promega) containing the DEVD motif. In the presence of active cas ...

129 This protocol is almost identical to that described in the previous section. The only difference being that this protocol ut ...

130 (c) 1 μL SP6 polymerase. (d) 1 μL amino acid mixture, minus methionine (20 μM fi nal concentration). (e) 2 μL [^35 S]methion ...

131 Supplement egg extract from Subheading 3.1 with 1:20 dilu- tion of 20× EM (and any treatments you wish you to exam- ine). I ...

132 and apoptosis. The protocol below is adapted from Nutt et al. (2005) [ 13 ]. In this study, we found that glucose-6-phosphat ...

133 Supplement egg extract from Subheading 3.1 with 1:20 dilu- tion of 20× EM. Divide this extract into two separate aliquots, ...

134 spontaneous apoptosis of X. laevis egg extracts was fi rst described [ 1 ]; however, it is not used as often today in favor ...

135 Place the oocytes in a 50-mL conical tube containing OR-2 buffer. Wash the oocytes several times with OR-2 buffer, removing ...

136 specialized equipment and knowledge of how to microinject oocytes, it is not diffi cult providing the equipment and tools ar ...

137 By lysing the eggs in the presence of EGTA, the effect of cal- cium will be blocked, and the eggs will remain arrested in m ...

138 It is best to prepare a “stock” egg extract supplemented with EM and then divide this between each sample you wish to assay ...

139 When injecting two solutions, they can be injected separately. However, care must be taken when injecting the same oocyte t ...

140 Deming P, Kornbluth S (2006) Study of apop- tosis in vitro using the Xenopus egg extract reconstitution system. Methods Mol ...

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

142 groups after the aspartate residue; (2) detection of the specifi c active caspase by western blot using an antibody specifi ...

143 Lysis buffer: 50 mM HEPES, pH 7.5, 0.1 % CHAPS, 2 mM dithiothreitol, 0.1 % Nonidet P-40, 1 mM EDTA, 1 mM phenylmethylsulfon ...

144 Stripping solution for western blot membranes: 62.5 mM Tris–HCl, pH 6.7, 2 % SDS, and 100 mM 2-mercaptoethanol (should be a ...

145 3 Methods Proteins susceptible to caspase cleavage possess a tetrapeptide recognition sequence containing the scissile P1 as ...

146 Mix well while avoiding bubbles and incubate the plate at 37 °C for 30 min and read the absorbance at 562 nm using a spectr ...

147 caspases has been previously performed in mouse tissues in cerebral ischemia [ 14 ], focal cerebral ischemia [ 16 ], lungs d ...