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Fig. 2. A typical chromatogram of IgGi purification using histidine li-
gand affinity adsorbent.
outlet from the W detector is connected to a fraction collector and
the fraction collector is set to collect fraction of -1 mL each.
5. Adjust the flow rate to a linear flow of 20 cm/h (-25 mL/h). Usually,
the whole setup is maintained at -+4”C either in a cold room or in a
special chromatographic chamber (see Note 8 for the temperature
variables and the choice of temperatures). Pump about 2-3 column
vol of the starting buffer through the column to equilibrate the gel
bed at the chosen pH and ionic concentration.
6. Ideally, pump through the column about 10 mg of the extract con-
taining the molecule to be separated (for example, IgGi subclass) in
a minimum vol of not more than 0.5 mL, equilibrated as described
above, at the same flow rate. Take care not to introduce any bubbles.
Then, continue to pump the equilibrating buffer (50 mMTris-HCI,
pH 7.4, in the case of IgGr purification), until no W absorbing mate-
rial comes out of the column. Then, start the elution with buffers
containing increasing amounts of NaCl(O.l-O.5M). Monitor the elu-
tion for W absorption and collect fractions for further analysis and
characterization. A typical chromatograph of IgGi purification from
a placental extract is represented in Fig. 2.
7. Verify the total recovery of proteins injected by the cumulative absor-
bance units of the fraction eluted with the different buffers. Any
strongly retained protein(s) should be removed before reusing the
same column. In the case of agarose based gels, this is usually done
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