Caspases,Paracaspases, and Metacaspases Methods and Protocols

228 Clear additional cell debris via centrifugation at 800 × g for 1 min at 4 °C. Transfer supernatant to chilled new microcent ...

229 Normalize volume of all samples and place on ice. A minimum volume of 200 μL is recommended. Assemble Bio-Dot SF apparatus ...

230 After staining, remove the membrane and place into the destain solution ( see Note 10 ). Wash the membrane on rocking platf ...

231 Discard supernatant and resuspend the beads in 1 mL of PBS containing 1 % (v/v) Triton-X 100 and wash for 10 min on nutator ...

232 Dissolve the FM dye to a stock concentration of 100 μM in 1× PBS, pH 7.4. Protect from light and place on ice. Add 20 μl of ...

233 Incubate at 4 °C for 12 h ( see Note 13 ). Discard supernatant and resuspend the cells in 1 mL of 50 mM sodium citrate. Col ...

234 protein fractions. For conducting normal immunoblots, prepare extracts using buffer A. For effi cient elution, ensure that ...

235 period with the stain. In order to avoid high background staining, additional washes may be performed as described in steps ...

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

238 While fungal and protozoan metacaspases have been impli- cated in a range of biological processes [ 2 , 4 ], the physiologic ...

239 of the same type. Yet extended specifi city of individual plant meta- caspases appears to be rather loose, as follows from s ...

240 Luria Broth (LB) medium: tryptone 10 g/L, yeast extract 5 g/L, NaCl 10 g/L. Add 15 g/L of Bacto Agar to solidify the medium ...

241 96-well fl at bottom tissue culture plates. Using black opaque plates will decrease the crosstalk and background noise duri ...

242 Radiolabeled L -[^35 S]methionine (PerkinElmer EasyTag™ Cat.NEG709A, or similar. RNasin ® Ribonuclease Inhibitor (Promega) ...

243 prolongation of induction time leads to accumulation of high amount of metacaspase in bacterial cells, this also induces int ...

244 Wash the column with several volumes of binding buffer. Wash the column with washing buffer for native conditions gradually ...

245 Although different metacaspases have many biochemical features in common (e.g., active as monomers and Ca 2+ -dependent), th ...

246 This approach is closer to in vivo assay and allows tracking activation or suppression of metacaspase activity in different ...

247 Dilute 5-μL aliquot of the supernatant ten times with Milli-Q or dH 2 O water and use it for estimating total protein conce ...