3 Methods
3.1 Ribosome
Preparation
1. After purification, buffer exchange 80S against Running buffer
with Amicon concentrator. We generally exchange step by step
a final volume of 15 mL Running buffer for an initial volume of
less than 1 mL of sample.
2. Determine the 80S concentration using a NanoDrop spectrophotometer. For an accurate concentration, perform three
independent twentieth dilutions of the stock solution (i.e.,
1 μL stock solution + 19 μL buffer), and measure the absorbance at 260 nm (ε 260 ¼ 50,000,000 M
À1 cm
À1
).
3. Dilute the stock solution with the 1Â Running buffer appropriately to make a maximal concentration of 2 μM in 500 μL.
Store at 4
C.
3.2 Designing
the switchSENSE
®
Experiment
3.2.1 Experimental
Considerations
DRX 2400 instrument was not originally designed for analysis of
complexes as large as ribosomes. Here, the size of the DNA probe
attached to the chip has been doubled, compared to standard
conditions, in order to deal with the large size of 80S ribosome.
Furthermore, measurements are done in “proximity sensing mode”
and not in “switching mode” (see Note 4) in order to avoid steric
constrains. Here we are taking advantage of an increase of fluorescence of the nanolever when the IRES RNA is bound by the
ribosome.
3.2.2 Experimental
Workflow Building
1. Open the switchBUILD software to design the experiment by
the creation of successive programming blocks.
2. In the first block, choose the biochip (MPC-96-2-Y1-S), channel number to be analyzed, Auxiliary Buffer (P40) and Running Buffer (Â140). Alternatively a custom buffer can be added
manually.
3. A “Passivation” step is added automatically as a second block
for all experiments (see Note 5).
4. To add a new measure block click “+” and select “Split Shot
Kinetics.”
5. In the new experimental panel in the “Properties” window,
select “Conjugate hybridization” in the immobilization
method and “static mode” in the measurement mode pulldown menus.
6. Input the name of ligand (CrPV IGR IRES—cNL-B96 oligonucleotide) and its concentration (see Note 6) and name of the
analyte (yeast ribosomes) and its mass.
7. To determine the experimental parameters for analyte injection, either press “PRESETS” and select an appropriate model
system or use the sliders for the different kinetic parameters:
344
Emma Schenckbecher et al.
3.1 Ribosome
Preparation
1. After purification, buffer exchange 80S against Running buffer
with Amicon concentrator. We generally exchange step by step
a final volume of 15 mL Running buffer for an initial volume of
less than 1 mL of sample.
2. Determine the 80S concentration using a NanoDrop spectrophotometer. For an accurate concentration, perform three
independent twentieth dilutions of the stock solution (i.e.,
1 μL stock solution + 19 μL buffer), and measure the absorbance at 260 nm (ε 260 ¼ 50,000,000 M
À1 cm
À1
).
3. Dilute the stock solution with the 1Â Running buffer appropriately to make a maximal concentration of 2 μM in 500 μL.
Store at 4
C.
3.2 Designing
the switchSENSE
®
Experiment
3.2.1 Experimental
Considerations
DRX 2400 instrument was not originally designed for analysis of
complexes as large as ribosomes. Here, the size of the DNA probe
attached to the chip has been doubled, compared to standard
conditions, in order to deal with the large size of 80S ribosome.
Furthermore, measurements are done in “proximity sensing mode”
and not in “switching mode” (see Note 4) in order to avoid steric
constrains. Here we are taking advantage of an increase of fluorescence of the nanolever when the IRES RNA is bound by the
ribosome.
3.2.2 Experimental
Workflow Building
1. Open the switchBUILD software to design the experiment by
the creation of successive programming blocks.
2. In the first block, choose the biochip (MPC-96-2-Y1-S), channel number to be analyzed, Auxiliary Buffer (P40) and Running Buffer (Â140). Alternatively a custom buffer can be added
manually.
3. A “Passivation” step is added automatically as a second block
for all experiments (see Note 5).
4. To add a new measure block click “+” and select “Split Shot
Kinetics.”
5. In the new experimental panel in the “Properties” window,
select “Conjugate hybridization” in the immobilization
method and “static mode” in the measurement mode pulldown menus.
6. Input the name of ligand (CrPV IGR IRES—cNL-B96 oligonucleotide) and its concentration (see Note 6) and name of the
analyte (yeast ribosomes) and its mass.
7. To determine the experimental parameters for analyte injection, either press “PRESETS” and select an appropriate model
system or use the sliders for the different kinetic parameters:
344
Emma Schenckbecher et al.