112
10. Wash the Ni
2+
-NTA agarose with at least six column volumes
of cold Ni
2+
-NTA wash buffer.
11. Elute the protein with 500 μL aliquots of Ni
2+
-NTA elution
buffer. Estimate the protein concentration of each fraction
using Bradford Reagent and/or absorbance at 280 nm.
12. Take aliquots of the eluate of the Ni
2+
-NTA column to follow and
analyze the purification process by SDS-PAGE (see Note 3).
13. Load all of the eluted fractions that contain protein onto a
StrepTactin-column.
14. Wash the column using the Strep-tag washing buffer. Wash
with six column volumes.
15. Elute the protein using 500 μL fractions of Strep-tag elution
buffer (for a 1 mL column). Estimate the protein concentration of each fraction using a Bradford quantification and/or
absorbance at 280 nm.
16. Take aliquots of each step and run an SDS-PAGE to verify the
purity of the protein.
17. Concentrate the protein using molecular-weight dependent
filtration devices, with the recommendation of the provider,
and change the buffer to storage buffer. Dilute the sample in a
1:1 ratio with glycerol 87% and store it at –20 °C at a concentration between 20 and 200 μM.
The synthesis of the intramolecular tether heavily depends on the
chemical properties of the intramolecular ligand. In one approach,
each component of the intramolecular tether (i.e., the BG-reactive
group, fluorophore, and intramolecular ligand) can be synthesized
separately in a modular approach and the tether subsequently
assembled using standard chemical methods such as amide bond
formation, click chemistry, and alkylation. For examples, see [22,
25, 27–30]. When making the intramolecular tether, it is worth
making also a ligand that only carries a fluorophore and can subsequently be used to measure the K d by means of fluorescence polarization measurements.
Many methods can be used to determine the affinity of the receptor protein for both the intramolecular ligand and the free analyte.
We routinely use fluorescence polarization to determine both of
these parameters.
1. Prepare serial dilutions of the receptor protein in labeling buffer that contains 20 nM fluorescein or tetramethylrhodamine
(TMR)-labeled ligand.
2. Pipette 100 μL of each dilution into the wells of a black 96-well
plate.
3. Incubate the plate for 10 min at room temperature.
3.2 Chemical
Synthesis
of the Intramolecular
Tether
3.3 Determining
the Affinity
of the Intramolecular
Ligand
Helen Farrants et al.
10. Wash the Ni
2+
-NTA agarose with at least six column volumes
of cold Ni
2+
-NTA wash buffer.
11. Elute the protein with 500 μL aliquots of Ni
2+
-NTA elution
buffer. Estimate the protein concentration of each fraction
using Bradford Reagent and/or absorbance at 280 nm.
12. Take aliquots of the eluate of the Ni
2+
-NTA column to follow and
analyze the purification process by SDS-PAGE (see Note 3).
13. Load all of the eluted fractions that contain protein onto a
StrepTactin-column.
14. Wash the column using the Strep-tag washing buffer. Wash
with six column volumes.
15. Elute the protein using 500 μL fractions of Strep-tag elution
buffer (for a 1 mL column). Estimate the protein concentration of each fraction using a Bradford quantification and/or
absorbance at 280 nm.
16. Take aliquots of each step and run an SDS-PAGE to verify the
purity of the protein.
17. Concentrate the protein using molecular-weight dependent
filtration devices, with the recommendation of the provider,
and change the buffer to storage buffer. Dilute the sample in a
1:1 ratio with glycerol 87% and store it at –20 °C at a concentration between 20 and 200 μM.
The synthesis of the intramolecular tether heavily depends on the
chemical properties of the intramolecular ligand. In one approach,
each component of the intramolecular tether (i.e., the BG-reactive
group, fluorophore, and intramolecular ligand) can be synthesized
separately in a modular approach and the tether subsequently
assembled using standard chemical methods such as amide bond
formation, click chemistry, and alkylation. For examples, see [22,
25, 27–30]. When making the intramolecular tether, it is worth
making also a ligand that only carries a fluorophore and can subsequently be used to measure the K d by means of fluorescence polarization measurements.
Many methods can be used to determine the affinity of the receptor protein for both the intramolecular ligand and the free analyte.
We routinely use fluorescence polarization to determine both of
these parameters.
1. Prepare serial dilutions of the receptor protein in labeling buffer that contains 20 nM fluorescein or tetramethylrhodamine
(TMR)-labeled ligand.
2. Pipette 100 μL of each dilution into the wells of a black 96-well
plate.
3. Incubate the plate for 10 min at room temperature.
3.2 Chemical
Synthesis
of the Intramolecular
Tether
3.3 Determining
the Affinity
of the Intramolecular
Ligand
Helen Farrants et al.