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5 M) can be used instead of ammonium sulphate. 8 M ammonium sulphate. Some monoclonal IgMs may bind too tightly to the column for complete elution in binding buffer. The remaining IgM will be eluted with wash buffer, but the high content of isopropanol will cause precipitation of IgM. Perform an immediate buffer exchange (see page 133) or dilute the sample to preserve the IgM. Lower concentrations of isopropanol may elute the IgM and decrease the risk of precipitation. To increase capacity, connect several HiTrap IgM Purification HP columns in series.

Coli periplasm containing Protein A-(HisGly)4His. Diluted with 9 ml binding buffer. 05 Mr 97 000 66 000 45 000 0 45 30 000 20 100 14 400 1 2 3 pool I 65 ml Lane 1. Low Molecular Weight Calibration Kit (LMW), reduced Lane 2. Crude periplasmic fraction, reduced Lane 3. Pool I, purified Protein A-(HisGly)4His, reduced Fig. 25. Purification of recombinant proteins on HiTrap Chelating HP, 5 ml, charged with Zn2+. Recombinant protein expressed in inclusion bodies Sample: 8 ml cell extract containing (His)10-tagged protein.

Equilibrate the column with 5 column volumes of binding buffer. 2. Apply the sample. 3. Wash with 5–10 column volumes of binding buffer. 4. Elute with 5–10 column volumes of elution buffer. 5. Wash with 5–10 column volumes of binding buffer. It is important to keep a low flow rate during sample loading and elution as the kinetics of the binding interaction between GST and glutathione are relatively slow. The binding capacity is protein dependent and therefore yield will vary according to the type of protein.

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