2.2.2 ISD and T3
Sequencing
1. MALDI steel plate.
2. Two matrix solutions: 25 mg/ml 1,5-diaminonaphthalene
(DAN) and 25 mg/ml of a mixture composed of
2,5-dihydroxybenzoic acid and 2-hydroxy-5-methoxybenzoic
acid (super-DHB). Both matrices are dissolved in 50% Acetonitrile (v/v), 0.1% (v/v) TFA in HPLC water.
3. ZipTip with C4 resin (Merck Millipore).
4. 1 mg/ml BSA in PBS as ISD standard.
2.3 Homogeneity
2.3.1 Dynamic Light
Scattering (DLS)
1. 0.22 μM Syringe filter.
2. Reusable quartz cuvettes (Hellma) or plates depending on the
model.
3. Detergent solution: 2% (v/v) Hellmanex.
4. Buffer used for PDZ sample: 1 ml.
5. PDZ sample.
2.3.2 Size Exclusion
Chromatography (SEC)
with Multi Angle Light
Scattering (SEC-MALS)
1. Size exclusion column (e.g., Superdex 75 increase from GE
healthcare).
2. PBS (137 mM [NaCl], 2.7 mM [KCl], 10 mM [Na2HPO4],
1.8 mM [KH2PO4]) (1 l) or other buffer (see Note 4).
3. PDZ sample. Prepare your sample in the chosen buffer with a
protein concentration at least of 1 mg/ml. This is particularly
true for PDZ domain if you want sufficient signal on the light
scattering detector as the signal is proportional to the molecular mass of the protein.
2.4 Conformational
Stability/Folding State
2.4.1 Circular
Dichroism (CD)
1. Aviv 215 spectropolarimeter.
2. 0.2 mm path-length cylindrical cell (Hellma) (see Note 5).
3. PDZ sample diluted at 0.2 mg/ml with PDZ sample buffer.
4. PDZ sample buffer.
5. 70% ethanol.
6. 2% (v/v) Hellmanex.
2.4.2 Differential
Scanning Fluorimetry
(Nano DSF)
1. Prometheus NT.48.
2. 70% ethanol to clean the lens.
3. Quartz capillary.
4. Buffer used for PDZ domain preparation and storage.
5. PDZ sample diluted at 0.2 mg/ml with PDZ sample buffer.
6. Peptide solubilized in PDZ sample buffer (tenfold molar excess
relative to PDZ sample).
104
Ce ´ lia Caillet-Saguy et al.
Sequencing
1. MALDI steel plate.
2. Two matrix solutions: 25 mg/ml 1,5-diaminonaphthalene
(DAN) and 25 mg/ml of a mixture composed of
2,5-dihydroxybenzoic acid and 2-hydroxy-5-methoxybenzoic
acid (super-DHB). Both matrices are dissolved in 50% Acetonitrile (v/v), 0.1% (v/v) TFA in HPLC water.
3. ZipTip with C4 resin (Merck Millipore).
4. 1 mg/ml BSA in PBS as ISD standard.
2.3 Homogeneity
2.3.1 Dynamic Light
Scattering (DLS)
1. 0.22 μM Syringe filter.
2. Reusable quartz cuvettes (Hellma) or plates depending on the
model.
3. Detergent solution: 2% (v/v) Hellmanex.
4. Buffer used for PDZ sample: 1 ml.
5. PDZ sample.
2.3.2 Size Exclusion
Chromatography (SEC)
with Multi Angle Light
Scattering (SEC-MALS)
1. Size exclusion column (e.g., Superdex 75 increase from GE
healthcare).
2. PBS (137 mM [NaCl], 2.7 mM [KCl], 10 mM [Na2HPO4],
1.8 mM [KH2PO4]) (1 l) or other buffer (see Note 4).
3. PDZ sample. Prepare your sample in the chosen buffer with a
protein concentration at least of 1 mg/ml. This is particularly
true for PDZ domain if you want sufficient signal on the light
scattering detector as the signal is proportional to the molecular mass of the protein.
2.4 Conformational
Stability/Folding State
2.4.1 Circular
Dichroism (CD)
1. Aviv 215 spectropolarimeter.
2. 0.2 mm path-length cylindrical cell (Hellma) (see Note 5).
3. PDZ sample diluted at 0.2 mg/ml with PDZ sample buffer.
4. PDZ sample buffer.
5. 70% ethanol.
6. 2% (v/v) Hellmanex.
2.4.2 Differential
Scanning Fluorimetry
(Nano DSF)
1. Prometheus NT.48.
2. 70% ethanol to clean the lens.
3. Quartz capillary.
4. Buffer used for PDZ domain preparation and storage.
5. PDZ sample diluted at 0.2 mg/ml with PDZ sample buffer.
6. Peptide solubilized in PDZ sample buffer (tenfold molar excess
relative to PDZ sample).
104
Ce ´ lia Caillet-Saguy et al.