207- ÄKTA liquid chromatography system (GE Healthcare) or
similar protein purifi cation system. - Bottle top vacuum fi lter (e.g., Nalgene reusable bottle top
fi lters). - Cellulose nitrate membrane fi lters (e.g., Whatman, 0.2 μm).
- Vacuum pump.
- 10 mL syringes and syringe-top fi lters (e.g., Millipore, 0.2 μm).
- Mechanical cell disrupter such as a French pressure cell or a
One-Shot cell disruptor (Constant Systems). - 2 mL and 20 mL centrifugal concentrators with a molecular
weight cutoff of 30 kDa (e.g., Vivaspin, Sartorius; Amicon
Ultra, Millipore). - 5 mL HisTrap Ni 2+ Sepharose (GE Healthcare) or equivalent
IMAC (immobilized metal affi nity chromatography) column. - HiLoad 16/60 Superdex 200 gel fi ltration column (S200
column, GE Healthcare). - Lysis Buffer: 20 mM sodium phosphate pH 7.5, 500 mM
NaCl, 0.1 mg/mL DNase, and 5 mM MgCl 2. - IMAC buffer A (Buffer A): 20 mM sodium phosphate pH 7.5
and 500 mM NaCl. - IMAC buffer B (Buffer B): 20 mM sodium phosphate pH 7.5,
500 mM NaCl, and 500 mM imidazole. - Gel fi ltration (GF) buffer: 10 mM Tris–HCl pH 7.0 and 5 mM
DTT (fi ltered and degassed).
A variety of materials can be used to exchange protein buffers.
Below is just a selection of the most commonly used.- Desalting columns (e.g., PD-10, GE Healthcare; Zeba Spin
columns, Pierce). - Dialysis tubing with a MWCO of 10–30 kDa (e.g., SnakeSkin
dialysis tubing, Pierce). - Centrifugal concentrators with MWCO of 30 kDa ( see Note 6 ).
- Size exclusion columns (e.g., S200 column, GE Healthcare).
- Spectrophotometer capable of absorbance measurements at
280 nm ( A 280 ). - Benchtop vortex mixer and microcentrifuge.
- Stereomicroscope for crystallography.
- Microcentrifuge tubes (0.5 mL and 1.8 mL).
- Liquid nitrogen.
- Crystallization incubator or temperature controlled room
(4 °C and ~21 °C).
2.4 Buffer Exchange
2.5 Crystallization,
Seeding, Cryoprotection,
and Heavy Atom Soaks
Trypanosoma Metacaspases