Caspases,Paracaspases, and Metacaspases Methods and Protocols

208 24-well sitting drop crystallization plates (e.g., Cryschem, Hampton). 96-well sitting drop crystallization plates (optiona ...

209 All inhibitors for testing (e.g., Z-VRPR-FMK) should be made up in 100 % DMSO or ddH 2 O at a concentration of 10–100 mM, de ...

210 Incubate the starter cultures for at least 8 h at 37 °C with constant shaking at ~225 rpm (the cultures can be left to grow ...

211 Prepare a 20 μL reduced sample by adding 5 μL sample buffer, 2 μL sample reducing agent, and 13 μL purifi ed protein (or a ...

212 Measure protein absorbance at 280 nm ( A 280 ) using a spectro- photometer and use this and the extinction coeffi cient (M ...

213 After a few seconds collect the crystal from the cryo-solution and immediately fl ash freeze by plunging into liquid nitrog ...

214 Consequently, the methods presented below may be useful for the structure solution of other Ca 2+ binding proteins where the ...

215 Read the fl uorescence using an excitation wavelength of 355 nm and emission wavelength of 460 nm. Fluorescence measurement ...

216 Choose a working protein concentration of around 5–10 μg/ mL or an optimized concentration from an earlier activity assay. ...

217 Add 100 μL ddH 2 O to 11 individual wells on single row (wells 1–11) of the 96-well plate. In well 12 add 150 μL of the sto ...

218 Exchange the protein into the Working Buffer and concen- trate to around 10 μM. Set up four 20 μL reactions in 1.8 mL micro ...

219 The expression vector pET28a+ (Novagen) was used for the protein expression of metacaspases from T. brucei. However, other ...

220 All fractions should be checked by SDS-PAGE to identify the protein, as the elution profi le may change depending on the te ...

221 References Coll NS, Vercammen D, Smidler A, Clover C, Van Breusegem F, Dangl JL, Epple P (2010) Arabidopsis type I metacasp ...

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

224 facilitate protein–protein interactions and contribute to the regulation of proteolytic activity [ 6 , 7 ]. Metacaspase func ...

225 The discovery of a well-entrenched non-death function of Yca1 has established metacaspases as versatile biological entities. ...

226 Vacuum source. Scotch Tape. YPD medium: 1 % (w/v) yeast extract, 2 % (w/v) Bacto Peptone, 2 % (v/v) dextrose, pH 3.5. FM li ...

227 Transfer between 5 and 10 μL of culture to fresh 50 mL of media. Incubate freshly inoculated 50 mL culture at 30 °C, 225 rp ...