Biomedical Engineering Reference
In-Depth Information
10.4.1.1
Specialized Materials
1. Labeled (radioactive or fl uorescent) in vitro transcribed or chemical synthesized
RNA.
2. Puri fi ed protein of interest.
3. Spermidine (100 mM) (Sigma-Aldrich).
4. RNAse OUT (Fermentas).
5. Heparin Sulfate (40 mg/mL) (Sigma-Aldrich).
Buffers
:
1 . 5 ×
Gel shift buffer
: 70 mM HEPES pH 7.9, 450 mM KCl, 11 mM MgCl
2
, 28%
glycerol.
2 .
Lysis buffer
: 50 mM HEPES pH 7.9, 150 mM KCl, 1 mM MgCl
2
, 1% Triton
X—100, 10% glycerol.
3 . 6 ×
Loading buffer
: 30% glycerol, 0.3% bromophenol blue, 0.3% xylene cyanol.
4. 10×
TBE
(
1 L
): 108 g tris base, 55 g boric acid, 9.3 g EDTA.
10.4.1.2
Methods
1. Prepare the following reaction in a microcentrifuge tube (final reaction volume is
14 mL): 1.5 mL of 1 mM spermidine (0.1 mM final concentration), 3 mL of 5× gel
shift buffer, 0.5 mL of RNase Inhibitor, X mL of Lysis buffer (as determined by
protein volume), 1 mL of radiolabeled RNA (we generally use ~5 fmoles;
50-100 K cpm/mL), and recombinant protein (100 nM).
2. Incubate at 30 °C for 5 min.
3. Add 1 mL of Heparin Sulfate (40 mg/mL) and transfer the reactions to ice for
5 min. This will reduce nonspecific interactions.
4. Add 3 mL 6× loading buffer. Load the reaction products on pre-run 5% non-
denaturing gel (native gel) at 200 V for 2-3 h to achieve good separation between
the input RNA and protein-RNA complexes.
5. Transfer the gel to Whatman filter paper, cover with plastic wrap, and dry with a
gel-drying apparatus.
6. The dried gel should be exposed to the phosphor screen and analyzed by
phosphorimaging.
10.4.1.3
Notes
1. The concentration of proteins and RNA must be known to calculate binding con-
stants. For a detailed description, see Ryder et al. (
2008
) .
2. RNA structure can strongly influence the migration of the transcript in native
gels. The addition of spermidine can help minimize effects of RNA structural
isoforms on migration of the transcript in the gel.
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