28
4 Notes
- Black plates are designed to reduce well-to-well cross talk and
background for fluorescence assays, whereas white plates are
designed to reduce well-to-well cross talk and background for
luminescence assays. White plates show higher fluorescence
background and amplify the fluorescence signal. Thus, more
accurate data can be obtained using black plates. - Stock solutions of peptidic substrates are prepared at 20 mM
to limit the final concentration of DMSO in enzymatic assays.
DMSO above 3 % can alter the catalytic activity of caspases. See
Subheading 3.2.1.1 for the preparation of Afc substrate
solutions. - Protease inhibitors can be added fresh to the lysis solution.
However, it is important that none of them affects the caspase
activity; thus, all-in-one tablets are not recommended. General
non-caspase protease inhibitors one can use are: 1,10-ortho-
phenanthroline (1 mM), 1 mM EDTA (already in the execu-
tioner caspase buffer), E-64 (10 μM), leupeptin (10 μM),
3,4-dichloroisocoumarin (10 μM), and MG-132 (1 μM). - The purity of the imidazole is very important. It must have a
low absorbance at 280 nm. Imidazole meeting ACS specifica-
tions is not suitable and will produce significant background
absorbance. The reagent needs to have 0.005 % β-NADH
equivalent as a fluorescence blank (e.g., Sigma cat. no. I-0250).
Some lower-grade imidazole alters the caspase activity. - The pET vectors are either ampicillin- (pET-15b/23b(+)) or
kanamycin- (pET-28b(+)) resistant, whereas the pLysS plasmid
confers resistance to chloramphenicol. Always use a freshly
transformed colony (1–2 days) to initiate the culture. Do not
keep cDNA in BL21 strains because of potential stability issues. - Sufficient yields of most caspases are obtained from BL21(DE3)
pLysS bacteria grown in rich medium such as 2× TY. However,
higher yields can be obtained using a richer medium (supple-
mented with 5 % glycerol or 2 % glucose) or a buffered medium
(Terrific Broth/TB medium). Those media generally result in
higher bacterial density, thus increasing yields. However, 2× TY
is much simpler to prepare and can be fully sterilized by auto-
claving. It is recommended to use baffled flasks instead of regu-
lar flasks because the former provide better aeration and can be
filled up to half the volume. Pre-heat the bacterial medium at
37 °C before starting your expression culture. This step ensures
that the optimal bacterial density is reached more quickly. - Not all caspases express well under these conditions. For
instance, more incubation time must be allowed for caspase-6
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