77- In vitro-coupled transcription/translation kit, such as the
TNT™ Coupled Reticulocyte Lysate System (commercially
available from Promega Corporation, USA). - 35 S-methionine (ICN Biochemicals), for the synthesis of radio-
labeled proteins. Labeled proteins can be stored for up to
2 weeks at −70 °C. - Ribonuclease inhibitor, such as RNasin (Ambion) or
RNAseOUT ribonuclease inhibitor (Invitrogen). - Heat block set to 30 °C.
- Gel precasting apparatus.
- Resolving gel: 10–15 % acrylamide/bis solution (37.5:1 with
2.6 % C), 375 mM Tris–HCl, pH 8.8, 0.1 % SDS, 0.1 % ammo-
nium persulfate (APS), 0.05 % N , N , N , N ′-Tetramethyl-
ethylenediamine (TEMED). - Stacking gel: 5 % acrylamide/bis solution (37.5:1 with 2.6 %
C), 125 mM Tris–HCl, pH 6.8, 0.1 % SDS, 0.1 % APS, 0.1 %
TEMED (note that acrylamide is a neurotoxin when unpo-
lymerized and so care should be taken to avoid exposure).
APS is prepared as a 10 % solution in water and frozen in ali-
quots at −20 °C. - Running Buffer (1×): 25 mM Tris-HCl, 192 mM glycine,
0.1 % SDS. A 5× buffer can be pre-made and stored at room
temperature. - Pre-stained molecular weight markers such as Kaleidoscope
markers (Bio-Rad), pre-stained protein markers (Invitrogen),
or unstained protein markers (Invitrogen). - Protein electrophoresis apparatus, such as Hoefer minigel tank
or Bio-Rad Mini-PROTEAN Electrophoresis system. - Power pack supply with constant voltage and constant current
settings. - Protein stain such as Coomassie Brilliant Blue R-250 (Bio-
Rad) made up as a 0.25 % (w/v) stock in H 2 O/methanol/
acetic acid (5:4:1). - Protein Transfer Buffer: 49 mM Tris-HCl pH 7.0, 39 mM gly-
cine, 0.0375 % SDS, 20 % methanol. - Polyvinylidene difl uoride (PVDF), such as low-fl uorescence
Immobilon-FL (Millipore). - Whatman fi lter paper.
- Protein transfer apparatus; either semidry transfer system
(Hoefer) or wet transfer system such as the Mini-Trans Blot
from Bio-Rad. - Rocking platform or shaker.
2.5 In Vitro
Translation of Caspase
Substrates
2.6 Protein
Electrophoresis
2.7 Immunoblotting
Assays for Studying Caspase-2