182
2. Plasmid pET29a_BLIP_NtermC (encoding periplasmic leader
sequence, His-tag, Thrombin cleavage site, cysteine, GGSHG
linker, β-lactamase inhibitor protein (BLIP)) (see Notes 1 and 2).
3. Competent Escherichia coli BL21 (DE3) cells.
4. LB medium: 10 g peptone, 5 g yeast extract and 10 g NaCl in
1 L dH 2 O supplemented with 30 mg/L kanamycin.
5. 100 mM isopropyl β-d-1-thiogalactopyranoside (IPTG) stock
solution. Filtered to sterilize (0.2 μm).
6. Shaking incubator.
7. High-speed centrifuge.
8. Osmotic shock solution: 30 mM Tris–HCl, 1 mM EDTA, 20%
sucrose at pH 8.0.
9. Low salt solution: 5 mM MgSO 4 .
10. 10× Tris–HCl buffer: 200 mM Tris–HCl at pH 7.4.
11. His-tag binding resin.
12. Charge buffer: 50 mM NiSO 4 .
13. Bind buffer: 20 mM Tris–HCl, 500 mM NaCl, 30 mM
imidazole at pH 7.9.
14. Wash buffer: 20 mM Tris–HCl, 500 mM NaCl, 60 mM imidazole
at pH 7.9.
15. Elution buffer: 20 mM Tris–HCl, 500 mM NaCl, 400 mM
imidazole, 2 mM TCEP at pH 7.9.
16. Storage buffer: 20 mM Tris–HCl, 150 mM NaCl, 2 mM
TCEP at pH 7.9.
17. Amicon centrifugal filter unit (NMWL = 10 kDa).
18. Nanodrop UV-VIS spectrophotometer.
1. Oligonucleotides modified with a primary amine at their 3′- or
5′-terminus (e.g., Integrated DNA Technologies).
2. Dimethylsulfoxide (DMSO).
3. Ethanol (cooled to −30 °C).
4. Phosphate buffered saline (PBS): 100 mM sodium phosphate,
150 mM NaCl at pH 7.2.
5. 5 M NaCl.
6. Sulfosuccinimidyl
4-(N-maleimidomethyl)cyclohexane-1carboxylate (Sulfo-SMCC, no-weigh format).
1. Ligation buffer: 100 mM sodium phosphate at pH 7.0.
2. Desalting column: PD-10 (e.g., GE Healthcare, Cat No:
17-0851-01).
3. Strong anion-exchange spin column, mini (e.g., Thermo
Scientific, Cat No: PI90008).
2.2 Maleimide
Functionalization
of Amine- Modified
Oligonucleotides
2.3 Conjugation
of MaleimideFunctionalized
Oligonucleotides
to Proteins
Wouter Engelen and Maarten Merkx
2. Plasmid pET29a_BLIP_NtermC (encoding periplasmic leader
sequence, His-tag, Thrombin cleavage site, cysteine, GGSHG
linker, β-lactamase inhibitor protein (BLIP)) (see Notes 1 and 2).
3. Competent Escherichia coli BL21 (DE3) cells.
4. LB medium: 10 g peptone, 5 g yeast extract and 10 g NaCl in
1 L dH 2 O supplemented with 30 mg/L kanamycin.
5. 100 mM isopropyl β-d-1-thiogalactopyranoside (IPTG) stock
solution. Filtered to sterilize (0.2 μm).
6. Shaking incubator.
7. High-speed centrifuge.
8. Osmotic shock solution: 30 mM Tris–HCl, 1 mM EDTA, 20%
sucrose at pH 8.0.
9. Low salt solution: 5 mM MgSO 4 .
10. 10× Tris–HCl buffer: 200 mM Tris–HCl at pH 7.4.
11. His-tag binding resin.
12. Charge buffer: 50 mM NiSO 4 .
13. Bind buffer: 20 mM Tris–HCl, 500 mM NaCl, 30 mM
imidazole at pH 7.9.
14. Wash buffer: 20 mM Tris–HCl, 500 mM NaCl, 60 mM imidazole
at pH 7.9.
15. Elution buffer: 20 mM Tris–HCl, 500 mM NaCl, 400 mM
imidazole, 2 mM TCEP at pH 7.9.
16. Storage buffer: 20 mM Tris–HCl, 150 mM NaCl, 2 mM
TCEP at pH 7.9.
17. Amicon centrifugal filter unit (NMWL = 10 kDa).
18. Nanodrop UV-VIS spectrophotometer.
1. Oligonucleotides modified with a primary amine at their 3′- or
5′-terminus (e.g., Integrated DNA Technologies).
2. Dimethylsulfoxide (DMSO).
3. Ethanol (cooled to −30 °C).
4. Phosphate buffered saline (PBS): 100 mM sodium phosphate,
150 mM NaCl at pH 7.2.
5. 5 M NaCl.
6. Sulfosuccinimidyl
4-(N-maleimidomethyl)cyclohexane-1carboxylate (Sulfo-SMCC, no-weigh format).
1. Ligation buffer: 100 mM sodium phosphate at pH 7.0.
2. Desalting column: PD-10 (e.g., GE Healthcare, Cat No:
17-0851-01).
3. Strong anion-exchange spin column, mini (e.g., Thermo
Scientific, Cat No: PI90008).
2.2 Maleimide
Functionalization
of Amine- Modified
Oligonucleotides
2.3 Conjugation
of MaleimideFunctionalized
Oligonucleotides
to Proteins
Wouter Engelen and Maarten Merkx