In this study, we describe an original use of this technology to
study kinetics binding of CrPV IGR IRES with the 80S yeast
ribosome. We showed that this method, originally dedicated to
smaller complexes, is also well adapted to the study of large macromolecular complexes.
2 Materials
2.1 Instruments
and Accessories
1. DRX 2400 or DRX
2 instrument (Dynamic Biosensors).
2. Nanodrop ND-1000 spectrophotometer (Thermo Fisher) or
equivalent.
3. switchCONTROL (version 1.6.20.2393), switchBUILD (version 1.12.0.21), and switchANALYSER (version 1.9.0.31)
software (Dynamic Biosensors).
4. Biochip for large complexes studies (MPC-96-2-Y1-S,
Dynamic Biosensors, see Note 1) bearing a 96-mer DNA
probe (nanolever) attached to the surface at the 5
0 end.
5. 1.5 and 10 mL autosampler vials with septa caps and insets.
2.2 Buffers
1. Passivation solution: as supplied by manufacturer (SOL-PAS1-5, Dynamic Biosensors).
2. Regeneration solution: as supplied by manufacturer (SOL-REG-12-1, Dynamic Biosensors).
3. 10Â Auxiliary Buffer: 100 mM sodium phosphate, pH 7.0,
400 mM NaCl, 0.5% (v/v) Tween 20, 0.5 mM EDTA.
4. Running buffer: 20 mM Na cacodylate, pH 7.0, 7 mM MgCl 2 ,
100 mM KCl, 0.05% (v/v) Tween 20 (see Note 2).
5. Ribosome buffer: 50 mM Na cacodylate, pH 7.0, 7 mM
MgCl 2 , 30 mM KCl, 70 mM NH 4 Cl, 1 mM DTT, 5% (w/v)
glycerol.
6. Ultrapure RNase-free water (homemade, used for the preparation of all buffers).
2.3 Ligand
Preparation
1. CrPV IGR IRES—cNL-B96 RNA sequence, complementary
to the 96-mer DNA probe onto the chip (see Note 3); stored at
1 mg/mL in 10 mM 2-(N-morpholino)ethanesulfonic acid,
200 mM NaCl at À20
C.
2.4 Analyte
Preparation
1. 80S S. cerevisiae ribosome purified from JD1370 strain following a protocol adapted from Ben Shem et al. ([17]; aliquots,
flash-frozen in liquid nitrogen at 10 μM and stored at À80
C
or freshly prepared, in Ribosome buffer).
2. Amicon Ultra-15 Centrifugal Filter with 100 kDa molecular
weight cutoff (EMD Millipore).
switchSENSE Analysis of 80S Ribosome/IRES Interactions
343
study kinetics binding of CrPV IGR IRES with the 80S yeast
ribosome. We showed that this method, originally dedicated to
smaller complexes, is also well adapted to the study of large macromolecular complexes.
2 Materials
2.1 Instruments
and Accessories
1. DRX 2400 or DRX
2 instrument (Dynamic Biosensors).
2. Nanodrop ND-1000 spectrophotometer (Thermo Fisher) or
equivalent.
3. switchCONTROL (version 1.6.20.2393), switchBUILD (version 1.12.0.21), and switchANALYSER (version 1.9.0.31)
software (Dynamic Biosensors).
4. Biochip for large complexes studies (MPC-96-2-Y1-S,
Dynamic Biosensors, see Note 1) bearing a 96-mer DNA
probe (nanolever) attached to the surface at the 5
0 end.
5. 1.5 and 10 mL autosampler vials with septa caps and insets.
2.2 Buffers
1. Passivation solution: as supplied by manufacturer (SOL-PAS1-5, Dynamic Biosensors).
2. Regeneration solution: as supplied by manufacturer (SOL-REG-12-1, Dynamic Biosensors).
3. 10Â Auxiliary Buffer: 100 mM sodium phosphate, pH 7.0,
400 mM NaCl, 0.5% (v/v) Tween 20, 0.5 mM EDTA.
4. Running buffer: 20 mM Na cacodylate, pH 7.0, 7 mM MgCl 2 ,
100 mM KCl, 0.05% (v/v) Tween 20 (see Note 2).
5. Ribosome buffer: 50 mM Na cacodylate, pH 7.0, 7 mM
MgCl 2 , 30 mM KCl, 70 mM NH 4 Cl, 1 mM DTT, 5% (w/v)
glycerol.
6. Ultrapure RNase-free water (homemade, used for the preparation of all buffers).
2.3 Ligand
Preparation
1. CrPV IGR IRES—cNL-B96 RNA sequence, complementary
to the 96-mer DNA probe onto the chip (see Note 3); stored at
1 mg/mL in 10 mM 2-(N-morpholino)ethanesulfonic acid,
200 mM NaCl at À20
C.
2.4 Analyte
Preparation
1. 80S S. cerevisiae ribosome purified from JD1370 strain following a protocol adapted from Ben Shem et al. ([17]; aliquots,
flash-frozen in liquid nitrogen at 10 μM and stored at À80
C
or freshly prepared, in Ribosome buffer).
2. Amicon Ultra-15 Centrifugal Filter with 100 kDa molecular
weight cutoff (EMD Millipore).
switchSENSE Analysis of 80S Ribosome/IRES Interactions
343