208
- 24-well sitting drop crystallization plates (e.g., Cryschem,
Hampton). - 96-well sitting drop crystallization plates (optional) (e.g.,
MRC crystallization plate, Molecular Dimensions). - Glass coverslip or microscope slide with depression.
- Sealant for crystallization trays (e.g., ClearVue Sheets,
Molecular Dimensions). - Adhesive PCR foil for sealing trays prior to crystallization
(optional) (Fischer Scientifi c). - Cryogenic loops (cryoloop), caps, and vials (e.g., LithoLoops,
CryoCaps, and CryoVials, Molecular dimensions). - Seed beads (e.g., Seed bead kit, Hampton Research; MicroSeed
beads, Molecular Dimensions). - Crystallization Solution: 50 mM Hepes pH 7.0, 0.1 % (w/v)
tryptone, and 20 % (w/v) PEG3350 ( see Note 7 ). - Sparse matrix crystallization screening kits (commercially
available from Hampton Research, Sigma, or homemade)
( see Note 8 ). - Cryoprotectant: 2-Methy-2-4-pentandiol (MPD).
- Samarium acetate (SmAc).
- Spectrophotometer capable of absorbance measurements at
280 nm ( A 280 ). - Fluorescence multi-well plate reader (e.g., Envision 2101,
PerkinElmer), equipped with excitation and emission fi lters of
355 nm and 460 nm respectively ( see Note 9 ). - Black, fl at-bottomed, 96-well plates.
- 1.8 mL microcentrifuge tubes.
- Data analysis software (e.g., GraFit, Erithacus Software or
Prism, GraphPad software). - Exchange Buffer (for buffer exchange): 50 mM Tris pH 7.5,
100 mM NaCl. - 1× Assay Buffer: 50 mM Tris–HCl pH 7.5, 100 mM NaCl,
5 mM DTT, 1 mM CaCl 2. - 2× Assay Buffer: 100 mM Tris pH 7.5, 200 mM NaCl, 10 mM
DTT, 2 mM CaCl 2 (allow for 100 μL per well). - 7-Amino-4-methylcoumarin (AMC) reference standard
(e.g., Sigma) made up to 20 mM in DMSO. - Commercial fl uorogenic substrates made up to 100 mM in
100 % DMSO, e.g., Z-GGR-AMC, Z-RR-AMC, Bz-R-AMC
(all Bachem) Z-FPR-AMC (MP Biomedicals), Z-VRPR-
AMC [ 18 ].
2.6 Metacaspase
Activity Assays
Karen McLuskey et al.http://www.ebook3000.com