Biology Reference
In-Depth Information
macromolecules. There seems to be an unusual interaction
between nucleic acids and the resin in the presence of thiol-
containing reducing reagents.
6. Heme absorbs at 280 nm. Thus, it is not surprising that
ε
280 nm
of the Fe(III) heme-NC1 complex (
ε
280 nm,holo
) is higher than
that of the protein alone calculated from the amino acid
sequence (
ε
280 nm,apo
).
7. The extinction coefficient for the 450 nm peak of NC1 is
74 mM
−1
cm
−1
[
30
]. This value was determined using the pyri-
dine hemochromagen assay [
37
] and should be used instead of
the previously reported value (58 mM
−1
cm
−1
) [
4
].
8. The heme occupancy (
O
heme
) may be calculated using the
following equation:
A
A
ε
×
450
280
,
apo
280
O
=
,
A
A
heme
(
)
×
ε
−
ε
−
ε
450
450
280
,
holo
280
,
apo
280
where the
ε
450 nm
of NC1 is 74 mM
−1
cm
−1
[
30
].
9. The above reaction can also be set up anaerobically using an
anaerobic chamber to prepare the protein sample and a gastight
syringe to transfer the pri-miRNA to the sample [
6
]. To do
kinetic assay, increase the above volumes and take aliquots at
desired intervals.
10. Polyacrylamide gels ≥15 % do not stick strongly to filter paper
typically used in gel drying protocols but adhere to the hydro-
phobic surface of the Gelbond film. Note that this surface is
opposite to the treated hydrophilic side that is designed to
cross-link with acrylamide.
11. There is a low level of nonspecific nuclease activity associated
with the His
6
-Drosha
390-1374
protein, which may originate from
either Drosha itself or residual impurities present in the Drosha
preparation. The pre-miRNA is usually the most stable among
the three specific Drosha cleavage products and thus is used for
quantification.
Acknowledgments
We thank Jen Cleveland for comments. This work was supported
by the National Institute of General Medical Sciences of the
National Institutes of Health under award number R01GM080563
(F.G.) and by a UCLA Dissertation Year Fellowship (I.B.).
