View - ResearchGate

Amylose Affinity Chromatography of MBP 177
2. Materials
2.1. Chemicals and Reagents
1. Inhibitor cocktail (see Note 7).
2. EGTA (see Note 8).
3. EDTA (see Note 8).
4. DTT.
5. Magnetic Separation Rack (New England BioLabs).
6. SDS.
7. Amylose magnetic beads (New England BioLabs).
8. Amylose agarose resin (New England BioLabs).
9. Amylose high flow resin (New England BioLabs).
10. Snakeskin Dialysis Membrane (10 kDa) (Pierce) or Slide-A-Lyzer 20 kDa
Cassette (Pierce).
11. Medical scalpel.
2.2. Amylose Affinity Chromatography at Small Scale Using
Magnetic Beads
1. Stationary support: Amylose magnetic beads (New England BioLabs).
2. Column preparation solution: 5% v/v methanol : ddH 2 O.
3. Column buffer: 50 mM N-2-Hydroxyethylpiperazine-N´-2-ethanesulfonic acid
(HEPES), 150 mM NaCl, 2 mM EDTA, 2 mM EGTA, 1 mM DTT pH 7.4 (see
Note 9).
4. Elution buffer: 50 mM HEPES, 150 mM NaCl, 1 mM DTT, 100 mM maltose
pH 7.4.
2.3. Calculation Experiment
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, 100 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.