Caspases,Paracaspases, and Metacaspases Methods and Protocols

188 Shaknovich R, Yang SN, Fang F, Gascoyne RD, Martinez-Climent JA, Glickman JF, Borden K, Wu H, Melnick A (2012) MALT1 small m ...

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

190 domain (containing the conserved catalytic dyad histidine and cysteine) fl anked by an N-terminal domain containing a mitoch ...

191 LmjMCA has been found to be an arginine-specifi c cysteine protease able to complement the yeast metacaspase (YCA1). In the ...

192 and z-GGR-AMC substrates, however, cd-LmjMCA activity with the Ac-VRPR-AMC substrate was increased when these two latter inh ...

193 L. major metacaspase gene: LmjF.35.1580 (Gene ID: 3684453). Metacaspase disrupted yeast cells: Euroscarf YCA1 disrupted str ...

194 Solution B: 3.67 ml H 2 O, 925 μl 10 M NaOH, 370 μl β-Mercaptoethanol, 50 μl 100 mM PMSF in isopropanol, 50 μl 0.5 M EDTA/K ...

195 The Bio-Rad Power Pac 3000 system or similar. Separating gel (12 %): mix 1.625 ml H 2 O, 2 ml 30 % acryl- amide–Bis-acrylam ...

196 Filter papers. Plastic wrap. Cassette and X-Ray fi lm. 3 Methods Plate Δyca1 cells ( see Note 1 ) from frozen stock onto ...

197 Inoculate one transformed colony into 1 ml of SD/DO/ Glucose medium and vortex. Transfer to 9 ml of SD/DO/Glucose medium an ...

198 Harvest transformed yeast cells from a 50 ml culture following 24 h of induction. The pellet can be kept frozen at –70 °C. ...

199 Wash 1 ml 50 % Ni-NTA resin with 2 ml of lysis buffer and add the supernatant (soluble proteins) to the washed resin. Incub ...

200 Incubate on the shaker at room temperature for 2 min in water to remove excess of Ponceau S red. Take a picture and mark mo ...

201 4 Notes Δyca1 cells can be obtained from Euroscarf Accession Number Y02453. ~10 ml overnight culture in 290 ml of YPD mediu ...

202 References Uren AG, O’Rourke K, Aravind LA, Pisabarro MT, Seshagiri S, Koonin EV, Dixit VM (2000) Identifi cation of paraca ...

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

204 Indeed, trypanosomatid metacaspases seemed to have evolved alternative functions, e.g., as a negative regulator of amastigot ...

205 binding studies were used to identify the calcium binding site, study potential TbMCA2 substrates, and identify Z-VRPR-FMK a ...

206 Prewarmed LB Agar plates: Dissolve 1 % (w/v) tryptone, 0.5 % (w/v) yeast extract, 1 % (w/v) NaCl, and 1.5 % (w/v) microagar ...

207 ÄKTA liquid chromatography system (GE Healthcare) or similar protein purifi cation system. Bottle top vacuum fi lter (e.g., ...