View - ResearchGate

178 Pattenden and Thomas
2.4. Amylose Affinity Chromatography in Batch and Semi-Batch
Modes Using Agarose and High Flow Matrices
1. Stationary support: Amylose agarose resin or high flow matrix (New England
BioLabs).
2. Column preparation solution: 5% v/v methanol : ddH 2 O.
3. Pre-equilibration buffer: 50 mM HEPES pH 7.4 (see Note 9).
4. Equilibration buffer: 50 mM HEPES, 150 mM NaCl, 2 mM EDTA, 2 mM EGTA,
1 mM DTT pH 7.4.
5. Wash buffer 1: 50 mM HEPES, 150 mM NaCl, 2 mM EDTA, 2 mM EGTA,
1 mM DTT, inhibitor cocktails pH 7.4.
6. Wash buffer 2: 50 mM HEPES, 150 mM NaCl, 1 mM DTT pH 7.4.
7. Elution buffer: 50 mM HEPES, 150 mM NaCl, 1 mM DTT, 50 mM maltose
pH 7.4.
8. Regeneration 1: 50 mM HEPES, 4 M Urea, 0.5% w/v SDS pH 7.4.
9. Regeneration 2: 50 mM HEPES, 150 mM (NH 4 ) 2 SO 4 , 2 mM EDTA, 2 mM
EGTA pH 7.4.
10. Regeneration 3: ddH 2 O.
11. Regeneration 4: 20% v/v ethanol : ddH 2 O.
2.5. FPLC Purification: Amylose High Flow Affinity Chromatography
1. Stationary support: Amylose high flow matrix (New England BioLabs).
2. Column preparation solution: 5% v/v methanol : ddH 2 O.
3. Pre-equilibration buffer: 50 mM HEPES pH 7.4 (see Note 9).
4. Column buffer: 50 mM HEPES, 150 mM NaCl, 1 mM DTT pH 7.4.
5. Elution buffer: 50 mM HEPES, 150 mM NaCl, 1 mM DTT, 20 mM maltose pH
7.4.
6. Regeneration 1: 50 mM HEPES, 4 M Urea, 0.5% w/v SDS pH 7.4.
7. Regeneration 2: 50 mM HEPES, 150 mM (NH 4 ) 2 SO 4 , 2 mM EDTA, 2 mM EGTA
pH 7.4.
8. Regeneration 3: ddH 2 O.
9. Regeneration 4: 20% v/v ethanol : ddH 2 O.
2.6. Matrix-Assisted Dialysis Refolding Methods
1. Stationary support: Amylose agarose resin or high flow matrix (New England
BioLabs).
2. Dialysis membrane, 10–30 kDa, or cassette.
3. Denaturation buffer: 50 mM HEPES, 6 M Urea, 1 mM DTT, 5 mM EDTA pH
7.5 (see Note 9).
4. Refold 1: 50 mM HEPES, 300 mM Urea, 150 mM NaCl, 1 mM DTT, 5 mM
EDTA pH 7.5.
5. Refold 2: 50 mM HEPES, 150 mM NaCl, 1 mM DTT, 5 mM EDTA pH 7.5.
6. Refold 3: 50 mM HEPES, 150 mM NaCl, 1 mM DTT pH 7.5.