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expression time for various caspases. Be consistent with the
expression time. A longer or a shorter expression period can
influence the activation and activity of the caspase and can con-
sequently affect results.- Even if proteins are purified immediately, a freeze–thaw cycle
will assist bacterial lysis. - The efficacy of lysis is somewhat difficult to assess. The lysate
will initially turn gooey as the bacterial DNA is released.
Sonication will break the DNA over time. The lysate will turn
from a thick, whole milk-like to a skim milk-like appearance.
For very large expressions, lysozyme can be added to a final
concentration of 1 mg/mL. Note that lysozyme is inhibited by
indole derivatives such as imidazole [ 46 ]. Therefore, do not
use a lysis buffer that includes imidazole, which is often used to
prevent binding of unwanted proteins on the IMAC resin. - The amount of resin varies based on the anticipated yield. As a
rule of thumb, 1 mL of resin will bind ~5 mg of caspase. It is
recommended to use just enough resin to bind the caspase and
not much more. However, if yield is low (<100 μg) and too
little purification resin is used, the E. coli SlyD protein, a
histidine- rich peptidyl prolyl cis-trans isomerase (UniProt
identification number P0A9K9) will compete with the His-
tagged protein for the resin. Consequently, do not use too lit-
tle resin (<0.5 mL). If pre-charged resin is used, omit step 4. - Lysate filtration helps to obtain a purer caspase preparation by
removing large floating debris. Durapore HV membranes have
a low protein-binding membrane, thus permitting more lysate
to be filtered before clogging. - If no gradient maker is available, step elution can be used (0 %,
10 %, 20 %, etc. ratio of elution buffer/bacterial lysis buffer).
However, gradient elution results in a preparation with a
higher degree of purity. A fast protein liquid chromatography
(FPLC) system can also be used. However, each run usually
takes more time than manual purification. Furthermore, the
utilization of an FPLC system does not allow for tailoring of
the amount of resin used and requires more buffer, which
increases cost; for example, high-grade imidazole is relatively
expensive. - To adequately visualize caspase subunits, the SDS-PAGE gel
system used must be carefully chosen. This is especially true for
caspases because the small subunit of caspases has an apparent
mobility of ~10 kDa. We recommend the use of gradient acryl-
amide gels using the ammediol buffer system, which is particu-
larly suited for the resolution of small proteins [ 47 ]. SDS- PAGE
analysis of the active caspase preparation will show two pro-
teins: a large subunit (~20 kDa plus N-terminal domain) and a
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