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252 Galaev and Mattiasson
3.4. Separation of T and B Lymphocytes Using Protein A-Cryogel
Monolithic Column (See Note 9)
1. Equilibrate protein A-cryogel column with 10 CV of 20 mM HEPES buffer, pH
7.4, containing 0.2 M NaCl (see Note 10).
2. Treat lymphocytes (1 ml, 2–4 × 10 7 cells/ml) with 50 μl (0.1 μg/μl) of goat antihuman
IgG(H+L) by incubating at 4°C for 15 min. Centrifuge the cells at 200 × g
for 10 min and re-suspend in 1 ml of balanced salt solution (see Note 11).
3. Apply the antibody-treated lymphocytes to the top of the column and let 1.5 ml of
liquid to flow through before allowing cells to run completely into the monolithic
column bed. Close the column outlet and allow cells to bind efficiently to the
matrix by incubating the column at room temperature for 10 min without any
buffer flow (see Note 12).
4. Apply 20 ml of HEPES buffer, pH 7.4, containing 0.2 M NaCl through the column
at a flow rate of 110 cm/h. Collect first 4 ml (see Note 13).
5. Apply 2 ml of dog IgG (30 mg/ml) to the column and incubate at 37°C for
1 h. Apply 4 ml more of dog IgG (30 mg/ml) and collect the eluted fraction
(see Note 14).
6. Regenerate the column with 10 CV of 0.1 M glycine–HCl buffer, pH 2.5,
containing 0.1 M NaCl at a flow rate of 110 cm/h. Store column at 4°C
(see Note 15).
7. Analyze the breakthrough and eluted fractions for the content of particular cell
lines (see Note 16).
4. Notes
1. General comments on protein purification using traditional IMAC adsorbents (see
Chapters 2 and 10) are applicable to the IMAC purification of histidine-tagged
proteins directly from crude extracts or fermentation broth using IMAC cryogels.
2. IDA-cryogel column washing and all the following steps are operated at a flow
rate of 12 ml/min (600 cm/h). This step is carried out to charge the column with
Cu(II) ions and washout all non-bound Cu(II) ions.
3. A small concentration of imidazole, 2 mM, in the running buffer favors washing
loosely bound Cu(II) ions and prevents non-specific binding of impurities to
Cu(II)-IDA ligands.
4. Do not exceed total load of 30 μg of His-tagged protein per 5 ml monolithic
IMAC-cryogel column. The feed loaded on the column could contain cell debris
or even the whole cells as the pores in the monolithic column are big enough
to allow for the free passage of particulate material through the column without
blocking the flow. Moreover, due to large pores, the flow resistance of the
column is very low allowing the use of flow rates as high as 600 cm/h without
deteriorating the column performance.
5. This step is carried out to wash cells and unbound soluble impurities. The cell
content is monitored by measuring absorbance at 450 nm.