2.2 Consumables
Two 96-well microplates are required for every assay with the Octet
RED96: a sample plate for the experiment and a plate for prehydrating the sensors. The sample volume for the plates is 200 μl per
well and for the Octet RED96 the plates must be Greiner catalogue
number 655209.
As noted above, a wide range of biosensors is available from
ForteBio (Sartorius group). For most of our work, we have used
streptavidin-coated biosensors to capture 5
0
biotinylated
oligonucleotides.
2.3 Reagents
1. Experimental buffer: The manufacturer’s recommended buffer
for routine measurements is PBS (or HBS) containing 0.1 mg/
ml BSA and 0.002% (v/v) Tween 20. The buffer should be the
same for all samples and in all experimental steps (see Note 2).
2. Biotinylated oligonucleotides: These can be purchased from
various suppliers (e.g., Dharmacon and Integrated DNA Technologies) (see Note 3).
3. Protein samples: These should be of the highest possible purity
and concentrations need to be accurately determined.
3 Methods
3.1 Standard Binding
Experiment
This section describes the method that is most often used in the
determination of equilibrium dissociation constants and kinetic
constants for the interaction of proteins with immobilized
oligonucleotides.
1. Soak the biosensors in experimental buffer using a sensor rack
and a 96-well microplate. The biosensors should be prehydrated for at least 20 min before initiating the measurement.
2. Fill three columns in the sample microplate with experimental
buffer.
3. Fill a column in the sample microplate with the biotinylated
oligonucleotide at the same concentration in each well (typically around 0.5 μg/ml) (see Note 4).
4. Fill a column in the sample microplate with protein at different
concentrations. Ideally, the concentration range should be
from 0.1 Â K d to 10 Â K d . In order to give adequate coverage
of this range, it is frequently necessary to repeat the measurement with a different set of protein concentrations.
5. The typical standard binding experiment involves five steps in
which sensors must be dipped into the different columns of the
96-well plate (see Fig. 1). Program the computer to perform
the following steps:
356
Stephen R. Martin et al.
Two 96-well microplates are required for every assay with the Octet
RED96: a sample plate for the experiment and a plate for prehydrating the sensors. The sample volume for the plates is 200 μl per
well and for the Octet RED96 the plates must be Greiner catalogue
number 655209.
As noted above, a wide range of biosensors is available from
ForteBio (Sartorius group). For most of our work, we have used
streptavidin-coated biosensors to capture 5
0
biotinylated
oligonucleotides.
2.3 Reagents
1. Experimental buffer: The manufacturer’s recommended buffer
for routine measurements is PBS (or HBS) containing 0.1 mg/
ml BSA and 0.002% (v/v) Tween 20. The buffer should be the
same for all samples and in all experimental steps (see Note 2).
2. Biotinylated oligonucleotides: These can be purchased from
various suppliers (e.g., Dharmacon and Integrated DNA Technologies) (see Note 3).
3. Protein samples: These should be of the highest possible purity
and concentrations need to be accurately determined.
3 Methods
3.1 Standard Binding
Experiment
This section describes the method that is most often used in the
determination of equilibrium dissociation constants and kinetic
constants for the interaction of proteins with immobilized
oligonucleotides.
1. Soak the biosensors in experimental buffer using a sensor rack
and a 96-well microplate. The biosensors should be prehydrated for at least 20 min before initiating the measurement.
2. Fill three columns in the sample microplate with experimental
buffer.
3. Fill a column in the sample microplate with the biotinylated
oligonucleotide at the same concentration in each well (typically around 0.5 μg/ml) (see Note 4).
4. Fill a column in the sample microplate with protein at different
concentrations. Ideally, the concentration range should be
from 0.1 Â K d to 10 Â K d . In order to give adequate coverage
of this range, it is frequently necessary to repeat the measurement with a different set of protein concentrations.
5. The typical standard binding experiment involves five steps in
which sensors must be dipped into the different columns of the
96-well plate (see Fig. 1). Program the computer to perform
the following steps:
356
Stephen R. Martin et al.