2. Luria–Bertani (LB) medium: 10 g/L tryptone, 5 g/L yeast
extract, and 10 g/L sodium chloride.
3. 100 mg/mL ampicillin (pET21a) or 50 mg/mL kanamycin
(pET28a) stock solution.
4. E. coli bacterial strain BL21(DE3) (Novagen).
5. 1 M isopropyl 1-thio-β-d-galactopyranoside (IPTG) stock
solution.
2.3 Reagents
and Solutions
All buffers should be of the highest purity available.
1. Binding buffer: 20 mM sodium phosphate pH 6.8, 50 mM
sodium chloride, and 0.5 mM ethylenediaminetetraacetic acid
(EDTA).
2. Peptides containing C-terminal PBMs are typically commercially synthesized. The peptides used here were chemically
synthesized by GenScript Inc. (Piscataway, NJ) and used at
>95% purity. Peptides corresponding to the C-terminus of
partner proteins were 8 amino acids long, N-terminally dansylated and contained a free carboxylate at the C-terminus. An
internal peptide from SH3-containing guanine nucleotide
exchange factor (SGEF) contained 14 amino acids and was
N-terminally dansylated. In addition, the C-terminal carboxylate group was amidated (see Note 3).
3. The amino acid sequence of the C-termini of the following
human proteins was used in the development of this protocol:
Neurexin-1 (residues 1470-1477: NKDKEYYV), Caspr4 (residues 1301-1308: ENQKEYFF), and Syndecan-1 (residues
303–310: TKQEEFYA). The internal SGEF peptide was
derived from residues 42–55: KPNGLLITDFPVED [18].
4. Resuspend ~2 mg aliquot of lyophilized peptide in 1 mL of
binding buffer and adjust the pH to 6.8 to obtain a highly
concentrated (1–2 mM) master stock solution.
5. Prepare a working stock solution of 0.130 mM stock
dansylated-PBM by diluting the master stock with binding
buffer (see Notes 4 and 5).
6. Store all PBM peptide stock solutions at À20
C in the dark (see
Note 6).
3 Methods
3.1 Purified CASK
and Scribble PDZ
Domains
A protocol for the expression of CASK and Scribble PDZ domains
is described below. Purification of these recombinant PDZ domains
is beyond the scope of this chapter but can be achieved using similar
methods and buffers as previously published [7, 8, 11]. In brief,
CASK PDZ was purified using cation exchange and size-exclusion
PDZ Domain Binding Thermodynamics
139
extract, and 10 g/L sodium chloride.
3. 100 mg/mL ampicillin (pET21a) or 50 mg/mL kanamycin
(pET28a) stock solution.
4. E. coli bacterial strain BL21(DE3) (Novagen).
5. 1 M isopropyl 1-thio-β-d-galactopyranoside (IPTG) stock
solution.
2.3 Reagents
and Solutions
All buffers should be of the highest purity available.
1. Binding buffer: 20 mM sodium phosphate pH 6.8, 50 mM
sodium chloride, and 0.5 mM ethylenediaminetetraacetic acid
(EDTA).
2. Peptides containing C-terminal PBMs are typically commercially synthesized. The peptides used here were chemically
synthesized by GenScript Inc. (Piscataway, NJ) and used at
>95% purity. Peptides corresponding to the C-terminus of
partner proteins were 8 amino acids long, N-terminally dansylated and contained a free carboxylate at the C-terminus. An
internal peptide from SH3-containing guanine nucleotide
exchange factor (SGEF) contained 14 amino acids and was
N-terminally dansylated. In addition, the C-terminal carboxylate group was amidated (see Note 3).
3. The amino acid sequence of the C-termini of the following
human proteins was used in the development of this protocol:
Neurexin-1 (residues 1470-1477: NKDKEYYV), Caspr4 (residues 1301-1308: ENQKEYFF), and Syndecan-1 (residues
303–310: TKQEEFYA). The internal SGEF peptide was
derived from residues 42–55: KPNGLLITDFPVED [18].
4. Resuspend ~2 mg aliquot of lyophilized peptide in 1 mL of
binding buffer and adjust the pH to 6.8 to obtain a highly
concentrated (1–2 mM) master stock solution.
5. Prepare a working stock solution of 0.130 mM stock
dansylated-PBM by diluting the master stock with binding
buffer (see Notes 4 and 5).
6. Store all PBM peptide stock solutions at À20
C in the dark (see
Note 6).
3 Methods
3.1 Purified CASK
and Scribble PDZ
Domains
A protocol for the expression of CASK and Scribble PDZ domains
is described below. Purification of these recombinant PDZ domains
is beyond the scope of this chapter but can be achieved using similar
methods and buffers as previously published [7, 8, 11]. In brief,
CASK PDZ was purified using cation exchange and size-exclusion
PDZ Domain Binding Thermodynamics
139