Biology Reference
In-Depth Information
Decade
RNA
ladder
LMWM
ladder
(nt)
150
pri-miR-30a
(150 nt)
5
′
3
′
Size
(nt)
100
100
90
90
80
80
pre-miR-30a
(63 nt)
70
70
60
60
50
3
′
product
(52 nt)
3
′
50
45
40
40
5
′
product
(35 nt)
5
′
35
30
30
1
23
Fig. 3
Example of a pri-miRNA processing assay. Uniformly labeled pri-miR-30a
was incubated with 4 nM His
6
-Drosha
390-1374
, either alone (
lane 2
) or with 50 nM
Fe(III) heme-bound NC1 dimer (
lane 3
), at 37 °C for 45 min. The reactions were
analyzed using a 7 M urea, 15 % polyacrylamide gel. LMWM: low molecular
weight marker
2. The TEV cleavage site in His
6
-Drosha
390-1374
allows the His
6
-
tag to be cleaved off using the TEV protease if desired but is
not used in the protocol presented here.
3. Measuring OD
600 nm
of cell cultures using a spectrophotometer
with a turbidity cuvette holder is more accurate than without,
because most of the scattered light is blocked by the turbidity
cuvette holder. The absolute values of OD
600 nm
depend on the
instrument used. The bacterial cell density we use in NC1
expression is similar to that commonly recommended for pro-
tein expression.
4. δ-ALA is a key heme biosynthesis intermediate. In the absence
of δ-ALA, NC1 is expressed as a mixture of heme-bound dimer
and heme-free “monomer” [
4
]. Addition of δ-ALA increases
the yield of NC1 expression and improves the heme content of
NC1 to the extent that often little or no heme-free “mono-
mer” is observed.
5. Presence of 1 mM DTT or 10 mM β-mercaptoethanol in the
SEC buffer helps remove the residual amount of nucleic acids
bound to NC1 during size exclusion chromatography using
the Superdex 200 column. Under this condition, the free
nucleic acids elute at >20 mL, a volume too large for
