chromatography (Superdex 75, GE Healthcare Life Sciences) while
purification of the first PDZ domain of Scribble was carried out
using nickel-chelate affinity chromatography. The N-terminal
6 Â His affinity tag of Scribble PDZ1 was removed by proteolysis
with recombinant tobacco etch virus (rTEV) protease for 36 h at
4
C. Undigested protein, cleaved 6 Â His tag, and His-tagged
rTEV were separated from the Scribble PDZ1 domain by nickelchelate chromatography. The digested PDZ1 protein was further
purified using Superdex 75 size-exclusion chromatography. All
proteins were concentrated to ~0.5–2.0 mM in binding buffer
(see Note 5). The prepared samples were generally used immediately (see Note 7).
1. Supplement LB medium with the appropriate antibiotic for the
desired PDZ domain (final concentration of 100 μg/mL ampicillin for the mpET21a CASK PDZ and 50 μg/mL kanamycin
for pET28a Scribble PDZ1).
2. Grow bacterial cells transformed with either CASK or Scribble
PDZ constructs in LB medium supplemented with antibiotic at
37
C under vigorous agitation to an optical density of a
0.6–0.8 measured at 600 nm wavelength.
3. Cool cultures to 18
C.
4. Induce protein expression by adding IPTG to 1 mM final
concentration.
5. Incubate for an additional 16–18 hrs at 18
C.
6. Harvest bacteria by centrifugation.
7. Proceed with the purification of proteins and concentrate to
~0.5 to 2.0 mM in binding buffer (see Note 5). The prepared
samples are generally used immediately (see Note 7).
3.2 Experimental
Sample Preparation
The experimental design calls for using serial dilutions of the PDZ
domain protein to cover the concentration range of 1–400 μM in
discrete steps. Thus, a 0.5 mM stock provides reliable quantification ranging from 1 to 100 μM K d , while a 2.0 mM stock provides
reliable quantification ranging from 20 to 200 μM K d . The PDZ
domain concentration range and number of titration points can be
adjusted in subsequent experiments to optimize the titration and
obtain a more reliable K d determination.
3.2.1 Cuvette and PBM
Peptide Preparation
1. Rinse the cuvette with distilled and deionized H 2 O (ddH 2 O)
and ethanol using a vacuum cuvette washer (see Note 8).
2. Air dry the cuvette after rinsing and/or cleaning.
3. Add stir bar and 1290 μL of binding buffer.
4. Add 10 μL of dansylated-PBM peptide stock solution to the
cuvette to obtain a 1.0 μM PBM peptide concentration (see
Note 2).
140
Young Joo Sun and Ernesto J. Fuentes
purification of the first PDZ domain of Scribble was carried out
using nickel-chelate affinity chromatography. The N-terminal
6 Â His affinity tag of Scribble PDZ1 was removed by proteolysis
with recombinant tobacco etch virus (rTEV) protease for 36 h at
4
C. Undigested protein, cleaved 6 Â His tag, and His-tagged
rTEV were separated from the Scribble PDZ1 domain by nickelchelate chromatography. The digested PDZ1 protein was further
purified using Superdex 75 size-exclusion chromatography. All
proteins were concentrated to ~0.5–2.0 mM in binding buffer
(see Note 5). The prepared samples were generally used immediately (see Note 7).
1. Supplement LB medium with the appropriate antibiotic for the
desired PDZ domain (final concentration of 100 μg/mL ampicillin for the mpET21a CASK PDZ and 50 μg/mL kanamycin
for pET28a Scribble PDZ1).
2. Grow bacterial cells transformed with either CASK or Scribble
PDZ constructs in LB medium supplemented with antibiotic at
37
C under vigorous agitation to an optical density of a
0.6–0.8 measured at 600 nm wavelength.
3. Cool cultures to 18
C.
4. Induce protein expression by adding IPTG to 1 mM final
concentration.
5. Incubate for an additional 16–18 hrs at 18
C.
6. Harvest bacteria by centrifugation.
7. Proceed with the purification of proteins and concentrate to
~0.5 to 2.0 mM in binding buffer (see Note 5). The prepared
samples are generally used immediately (see Note 7).
3.2 Experimental
Sample Preparation
The experimental design calls for using serial dilutions of the PDZ
domain protein to cover the concentration range of 1–400 μM in
discrete steps. Thus, a 0.5 mM stock provides reliable quantification ranging from 1 to 100 μM K d , while a 2.0 mM stock provides
reliable quantification ranging from 20 to 200 μM K d . The PDZ
domain concentration range and number of titration points can be
adjusted in subsequent experiments to optimize the titration and
obtain a more reliable K d determination.
3.2.1 Cuvette and PBM
Peptide Preparation
1. Rinse the cuvette with distilled and deionized H 2 O (ddH 2 O)
and ethanol using a vacuum cuvette washer (see Note 8).
2. Air dry the cuvette after rinsing and/or cleaning.
3. Add stir bar and 1290 μL of binding buffer.
4. Add 10 μL of dansylated-PBM peptide stock solution to the
cuvette to obtain a 1.0 μM PBM peptide concentration (see
Note 2).
140
Young Joo Sun and Ernesto J. Fuentes