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250 Galaev and Mattiasson
2.4. Separation of T and B Lymphocytes Using Protein A-Cryogel
Monolithic Column (See Note 9)
1. Running buffer: 20 mM HEPES buffer, pH 7.4, containing 0.2 M NaCl.
2. Elution buffer: Dog IgG (30 mg/ml) in 20 mM HEPES buffer, pH 7.4, containing
0.2 M NaCl.
3. Regeneration buffer: 0.1 M glycine–HCl buffer, pH 2.5, containing 0.1 M NaCl.
4. Feed: Lymphocytes are isolated from freshly collected human buffy coat using
Ficoll-Paque. The buffy coat (20 ml) is diluted with an equal volume of balanced
salt solution (0.145 M Tris–HCl, pH 7.6, containing 0.1% glucose, 0.05 mM
CaCl 2 , 0.98 mM MgCl 2 , 5.4 mM KCl and 14 mM NaCl). Six milliliters of
the diluted buffy coat is overlayed on 5-ml Ficoll-Paque in 15-ml tissue culture
plastic tube and then centrifuged at 400 × g for 40 min at room temperature. The
lymphocytes are collected at the interface. To minimize the contamination of red
blood cells, the lymphocytes collected in the above procedure are re-centrifuged
on Ficoll-Paque as above. The cells are washed twice with 10 ml of balanced
salt solution and centrifuged at 200 × g for 10 min. The washed lymphocytes
are then suspended in balanced salt solution and used within 24 h for further
experiments. Stationary support: 2 ml monolithic pre-activated cryogel produced
from polyDMAAm (Protista Biotechnology AB).
3. Methods
3.1. Coupling IMAC Ligand: Iminodiacetic Acid
1. Pass 50 ml 0.5 M Na 2 CO 3 followed by 50 ml 1MNa 2 CO 3 solutions through the
column at a flow rate of 75 cm/h.
2. Recycle 0.5 M IDA solution in 1MNa 2 CO 3 , pH 10, for 24 h at room temperature
through the column at a flow rate of 75 cm/h.
3. Wash the modified cryogel in the column with 0.5 M Na 2 CO 3 (100 ml) and then
with water until pH is around neutrality.
4. Load the IDA-cryogel with Cu(II) ions by passing 50 ml 0.5 M CuSO 4 (dissolved
in distilled water) through the column at flow rate of 75 cm/h.
5. Determine the amount of immobilized IDA for IDA-cryogel by assaying the
amount of bound copper ions at saturation assuming a stoichiometric ratio after
the adsorbent is saturated with Cu(II) ions. Elute the Cu(II) ions from the column
with 0.1 M EDTA, pH 7.6, and determine spectrophotometrically as absorbance
of Cu(II) complex formed in 0.1 M EDTA solution, pH 7.6 at max = 730 with
730 = 46.8 M/cm.
6. After elution, wash the IDA-cryogel column with 100 ml water at a flow rate of
75 cm/h and then dry at 60°C overnight.
7. Insert a dry IDA-cryogel column in Pharmacia chromatographic column (i.d. of
1 cm) supplied with adapters (or any other suitable column with i.d. of 1 cm).
8. Re-swell the IDA-cryogel column in the running buffer and adjust the ends of the
IDA-cryogel monolith.