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
13 μL sample containing purifi ed protein plus ddH 2 O) in a
microcentrifuge tube. - Heat samples for 10 min at 70 °C or for 3 min at 90 °C.
- Assemble mini-gel equipment and add 200 mL of MOPS or
MES Buffer to the upper chamber and 600 mL to the lower
chamber. - Add 200 μL of general reducing agent to the upper chamber.
- Load ~8 μL of the protein molecular weight marker and all of
the denatured protein samples (20 μL) on to the gel. - Run at 200 V for around 40 (MES Buffer) to 60 (MOPS
Buffer) min. - Remove gel from tank and wash three times (5 min) in water
with shaking. - Add Coomassie stain to the gel and leave shaking for ~1 h.
- Dispose of the staining solution and destain the gel with
ddH 2 O or appropriate destaining solution for ~1 h or until the
gel background is clear ( see Note 15 ).
Crystallization of active recombinant TbMCA2 has so far proved
unsuccessful. This may be a consequence of the mixed products
resulting from the enzyme exhibiting auto-processing during over-
expression in E. coli. To combat this, the catalytically inactive
mutant TbMCA2 C213A was used in crystallization experiments and
was found to be stable and routinely produce crystals. Unfortunately,
many of the initial crystals were composed of multiple plates that
were unsuitable for diffraction experiments. However, optimizing
them in 24-well sitting drop plates using microseeding techniques
proved very useful in obtaining diffraction-quality crystals (Fig. 1 )
and is highly recommended if the initial crystals grow in clusters.3.3 SDS-PAGE
Analysis
3.4 Crystallization
and Crystal Handling
Fig. 1 TbMCA2 C213A crystallization experiments. ( a ) Initial clusters of crystals were unsuitable for diffraction
experiments. ( b ) Improved singular crystals were obtained using microseeding techniques
Trypanosoma Metacaspases