dissociation constant K d ; association rate constant k on ; and
dissociation rate constant k off . Alternatively, the parameter
values can be directly entered into the text boxes.
8. Press “Auto Generate” and the highest starting analyte concentration, number of dilutions, association/dissociation
times, and flow rates will be automatically filled with the appropriate values determined by the program (see Note 7). These
values can be altered manually, for example, by increasing the
time for dissociation or increasing the number of concentrations tested. The graph of predicted results automatically
updates. In the specific case of our experiment, we chose the
following parameters: 100, 33, and 11 nM for ribosome concentration, 5 min at 50 μL/min for the association step, and
20 min at the same rate for dissociation.
9. Input the analysis temperature; select the electrode number to
be measured for both association and dissociation steps (one of
the six available in each channel). Electrode 3 at 25
C was
chosen for the experiment.
10. The regeneration step button must be checked (default value),
after each concentration for the association part, and chose the
dissociation event only at the last concentration (see Note 8).
11. It is highly recommended to perform blank runs using Running Buffer for both association and dissociation steps. To do
this, check that the “with blank run” button is checked for
both steps.
Finally, a “Standby” block is dragged into sequence at the
end of the experiment with the “Reset surface button” checked
(see Note 9).
12. Save the switchBUILD assay file in an appropriate folder but
keep the program open.
3.3 Performing
the switchSENSE
Experiment
1. Open the “Autosampler” window in the “Kinetics” block of
the switchBUILD experiment file. This displays the volumes,
concentrations, and positions in the autosampler rack, of all the
solutions required for the experiment (see Note 7). In our
example, three concentrations and buffer (as a blank run) are
tested (100, 33, 11, and 0 nM of ribosome in 250 μL of 1Â
Running buffer) at a flow of 50 μL/min after hybridization
with a solution of CrPV IGR IRES—cNL-B96 RNA at 380 nM
in 120 μL.
2. Fill the positions in autosampler as displayed with ligand, analyte, Ultrapure RNase-free water, Passivation, and Regeneration solutions and an empty vial for waste.
3. Dilute the Auxiliary Buffer 10Â tenfold with Ultrapure
RNA-free water: 20 mL stock plus 180 mL water. Insert the
appropriate inlet pipes into the 1Â Auxiliary and Running
buffers.
switchSENSE Analysis of 80S Ribosome/IRES Interactions
345
dissociation rate constant k off . Alternatively, the parameter
values can be directly entered into the text boxes.
8. Press “Auto Generate” and the highest starting analyte concentration, number of dilutions, association/dissociation
times, and flow rates will be automatically filled with the appropriate values determined by the program (see Note 7). These
values can be altered manually, for example, by increasing the
time for dissociation or increasing the number of concentrations tested. The graph of predicted results automatically
updates. In the specific case of our experiment, we chose the
following parameters: 100, 33, and 11 nM for ribosome concentration, 5 min at 50 μL/min for the association step, and
20 min at the same rate for dissociation.
9. Input the analysis temperature; select the electrode number to
be measured for both association and dissociation steps (one of
the six available in each channel). Electrode 3 at 25
C was
chosen for the experiment.
10. The regeneration step button must be checked (default value),
after each concentration for the association part, and chose the
dissociation event only at the last concentration (see Note 8).
11. It is highly recommended to perform blank runs using Running Buffer for both association and dissociation steps. To do
this, check that the “with blank run” button is checked for
both steps.
Finally, a “Standby” block is dragged into sequence at the
end of the experiment with the “Reset surface button” checked
(see Note 9).
12. Save the switchBUILD assay file in an appropriate folder but
keep the program open.
3.3 Performing
the switchSENSE
Experiment
1. Open the “Autosampler” window in the “Kinetics” block of
the switchBUILD experiment file. This displays the volumes,
concentrations, and positions in the autosampler rack, of all the
solutions required for the experiment (see Note 7). In our
example, three concentrations and buffer (as a blank run) are
tested (100, 33, 11, and 0 nM of ribosome in 250 μL of 1Â
Running buffer) at a flow of 50 μL/min after hybridization
with a solution of CrPV IGR IRES—cNL-B96 RNA at 380 nM
in 120 μL.
2. Fill the positions in autosampler as displayed with ligand, analyte, Ultrapure RNase-free water, Passivation, and Regeneration solutions and an empty vial for waste.
3. Dilute the Auxiliary Buffer 10Â tenfold with Ultrapure
RNA-free water: 20 mL stock plus 180 mL water. Insert the
appropriate inlet pipes into the 1Â Auxiliary and Running
buffers.
switchSENSE Analysis of 80S Ribosome/IRES Interactions
345