optimization for expression inSpodoptera frugiperda(Sf-9)
insect cells (seeNote 3).- Expression media and reagents: LB broth, LB agar plates,
100 mg/ml ampicillin, and 100 mM isopropylβ-D-thiogalac-
topyranoside (IPTG). - DH10BAC cells:E. colicells that contain bacmid DNA.
- Host insect cells:Spodoptera frugiperda(Sf-9) insect cells.
- Recombinant virus: Virus generated by transfecting insect cells
with recombinant bacmid DNA that contains the gene of
interest integrated into viral DNA. - Cell lysis buffer: 50 mM Tris–HCl, pH 8.0, 150 mM NaCl, 5%
(v/v) glycerol, 2 mM tris-2-carboxyethyl phosphine (TCEP),
20 mM imidazole, 0.001% (v/v) Triton X-100. - Cell sonicator: e.g., Branson ultrasonic disintegrator.
- Nickel affinity chromatography column: e.g., 5 ml HisTrap™
HP column. - Elution buffer: 50 mM Tris–HCl, pH 8.0, 150 mM NaCl, 5%
(v/v) glycerol, 2 mM TCEP, 20 mM imidazole, 0.001% (v/v)
Triton X-100, 300 mM imidazole. - Dialysis buffer: 50 mM Tris–HCl, pH 8.0, 150 mM NaCl, 5%
(v/v) glycerol, 2 mM TCEP, 0.001% (v/v) Triton X-100. - Size exclusion chromatography column: e.g., Superdex
200 HiLoad 16/60 column. - Size exclusion chromatography (SEC) buffer: 50 mM
Tris–HCl, pH 8.0, 150 mM NaCl, 5% (v/v) glycerol, 2 mM
TCEP, and 0.001% (v/v) Triton X-100. - Vector for expression in insect cells: e.g., pFastBac-1 insect cell
expression vector. - Serum-free insect cell medium: e.g., Sf-900 III SFM.
2.2 Hydrogen/
Deuterium Exchange
Coupled to Mass
Spectrometry
- Protein storage buffer: 50 mM Tris–HCl, pH 8.0, 150 mM
NaCl, 10% (v/v) glycerol, 2 mM TCEP, and 0.001% (v/v)
Triton X-100 (seeNote 4). - H 2 O HDX buffer: 50 mM Tris–HCl, pH 8.0, 150 mM NaCl,
10% (v/v) glycerol, 2 mM TCEP in H 2 O(seeNote 5). - D 2 O HDX or labeling buffer: 50 mM Tris–HCl, pH 8.0,
150 mM NaCl, 10% (v/v) glycerol, 2 mM TCEP in D 2 O(see
Notes 5and 6 ). - Quench buffer: 3 M urea, 1% (v/v) trifluoroacetic acid (TFA)
in H 2 O, pH 2.4 (seeNotes 7and 8 ). Add fresh reducing agent
(2 mM TCEP). - HDX Mobile phase A: 0.3% (v/v) formic acid in H 2 O.
Biophysical Studies to Evaluate Protein-Ligand Interactions 33