3.1 Labeling
the Receptor
1. Thaw one tube of Cyanine-5 NHS on ice. Add 9 μL of 1Â PBS
and mix by pipetting up and down (see Note 8). There may be
signs of dye precipitation at this point; they can be safely
disregarded.
2. Take 1 μL of the diluted dye and add it to the 200 μL of 20 μM
α-CT. Mix gently by pipetting up and down. Incubate at room
temperature for 30 min in the dark. During the last 10 min of
the incubation period, transfer the tube to a room-temperature
microcentrifuge and centrifuge it at maximum speed (usually
about 15,000 Â g) for 10 min.
3. While the labeling reaction incubates, equilibrate the smallscale desalting column with 9 mL of 1Â PBS by allowing the
solution to flow through the column by gravity.
4. Carefully remove the labeling reaction from the microcentrifuge and apply 190 μL of the supernatant to the top of the
equilibrated column, allowing the solution to fully enter the
column’s bed. Next, apply 210 μL of 1Â PBS, and allow it to
enter the bed also.
5. Situate a 1.5-mL microcentrifuge tube under the column, and
then apply 600 μL of 1Â PBS to the column, collecting all
eluent. Gently mix the eluent.
6. Using a spectrophotometer, scan the eluent from 250 to
800 nm (no dilution is necessary; see Note 9). Determine the
α-CT* (i.e., the concentration of the labeled protein) and the
efficiency of the labeling (ϕ) using the following formulas:
αÀCT
∗
¼
A 280 À 0:03A 640
ε 280 Â d
ϕ ¼
A 640
ε 640  d  αÀCT
∗
ð5Þ
where ε 640 is 250,000 M
À1 cm
À1 . Starting from a 20-μM stock,
we usually obtain 4–6 μM of the protein; using these conditions with α-CT, we obtained a final concentration of 4.4 μM
labeled at 13% efficiency (see Note 10).
7. Make 1 mL of a 100 nM working stock of the labeled protein
(see Note 11) by diluting the appropriate volume with 1Â PBS,
and make the final Tween-20 concentration of this solution
0.05% (v/v).
3.2 Optimizing
Capillaries, LED Power,
and Buffer Conditions
1. Mix 50 μL of the α-CT* working stock and 50 μL of PBS–
Tween (this is the “0 μM SBTI” sample). In a second tube, mix
50 μL of the α-CT* working stock and 50 μL of 40 μM SBTI
(this is the “20 μM SBTI” sample). Allow both mixtures to
incubate in the dark at room temperature for 30 min.
2. Fill three Standard and three Premium capillary tubes by dipping one of each kind of capillary into the 0 μM SBTI sample.
166
Shih-Chia Tso and Chad A. Brautigam
the Receptor
1. Thaw one tube of Cyanine-5 NHS on ice. Add 9 μL of 1Â PBS
and mix by pipetting up and down (see Note 8). There may be
signs of dye precipitation at this point; they can be safely
disregarded.
2. Take 1 μL of the diluted dye and add it to the 200 μL of 20 μM
α-CT. Mix gently by pipetting up and down. Incubate at room
temperature for 30 min in the dark. During the last 10 min of
the incubation period, transfer the tube to a room-temperature
microcentrifuge and centrifuge it at maximum speed (usually
about 15,000 Â g) for 10 min.
3. While the labeling reaction incubates, equilibrate the smallscale desalting column with 9 mL of 1Â PBS by allowing the
solution to flow through the column by gravity.
4. Carefully remove the labeling reaction from the microcentrifuge and apply 190 μL of the supernatant to the top of the
equilibrated column, allowing the solution to fully enter the
column’s bed. Next, apply 210 μL of 1Â PBS, and allow it to
enter the bed also.
5. Situate a 1.5-mL microcentrifuge tube under the column, and
then apply 600 μL of 1Â PBS to the column, collecting all
eluent. Gently mix the eluent.
6. Using a spectrophotometer, scan the eluent from 250 to
800 nm (no dilution is necessary; see Note 9). Determine the
α-CT* (i.e., the concentration of the labeled protein) and the
efficiency of the labeling (ϕ) using the following formulas:
αÀCT
∗
¼
A 280 À 0:03A 640
ε 280 Â d
ϕ ¼
A 640
ε 640  d  αÀCT
∗
ð5Þ
where ε 640 is 250,000 M
À1 cm
À1 . Starting from a 20-μM stock,
we usually obtain 4–6 μM of the protein; using these conditions with α-CT, we obtained a final concentration of 4.4 μM
labeled at 13% efficiency (see Note 10).
7. Make 1 mL of a 100 nM working stock of the labeled protein
(see Note 11) by diluting the appropriate volume with 1Â PBS,
and make the final Tween-20 concentration of this solution
0.05% (v/v).
3.2 Optimizing
Capillaries, LED Power,
and Buffer Conditions
1. Mix 50 μL of the α-CT* working stock and 50 μL of PBS–
Tween (this is the “0 μM SBTI” sample). In a second tube, mix
50 μL of the α-CT* working stock and 50 μL of 40 μM SBTI
(this is the “20 μM SBTI” sample). Allow both mixtures to
incubate in the dark at room temperature for 30 min.
2. Fill three Standard and three Premium capillary tubes by dipping one of each kind of capillary into the 0 μM SBTI sample.
166
Shih-Chia Tso and Chad A. Brautigam