107- Stain the membrane with the Ponceau S solution at room tem-
perature for 5 min and wash with destaining buffer for 10 min. - Wash the membrane twice with distilled water for 5 min each.
- Excise the predicted size of the stained protein band and use the
membrane strip for N-terminal microsequencing ( see Note 12 ).
4 Notes
- Any pET vector carrying a six-histidine affi nity tag can be
used. - A His 6 affi nity tag or a FLAG affi nity tag can be used for the
purifi cation. But based on our experience, the activity of
CED-3 purifi ed using a FLAG tag was lower than that purifi ed
with a His 6 tag. The difference may be due to the concentra-
tion of elution in each eluting fraction. CED-3 with a His 6 tag
can be eluted in high concentrations by controlling the con-
centrations of imidazole, while it is more diffi cult to do so with
FLAG peptides. - CED-3 zymogen is auto-processed to generate 24, 17, and
13 kDa fragments [ 18 ]. Two of the fragments, 17 and 13 kDa,
form a heterodimer that constitutes the catalytic protease and
are positioned at the middle and at the C-terminus of the
zymogen, respectively. Therefore, a C-terminal His 6 tag should
be used for purifying CED-3 protease. - A micro-tip can also be used, but a fl at tip is more effi cient in
this purifi cation process, because the volume of CED-3 lysate
is large. - For the detection of fl uorogenic substrates, 96-well plates
designed for UV light should be used. - Once the pellet has been frozen, it can be stored at −80 °C for
up to several weeks. - The recommended procedure for sonication is 2 s on, 10 s off,
and with sonicator at 40 % amplitude in a 4 °C room using a
fl at tip for a total of 5 min. - Do not allow the resin to dry, even for a short period of time.
- Generally the highest concentration of CED-3 is eluted in elu-
tion buffer 2. Sometimes, a higher concentration of imidazole
(1 M) may be applied to the column for the fi nal elution. - Fluorogenic substrate stock solution can be made at 1 mM
concentration in DMSO and stored at −20 °C. - Promega’s manual recommends a 50 μl reaction. However, we
found that a 10 μl reaction usually provides enough labeled
product for the CED-3 cleavage assay. - At least 0.5–1 μg protein fragments are needed for successful
microsequencing.
In Vitro CED-3 Assays