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Extra info for Affinity Chromatography
8 M ammonium sulphate. 0 M. To avoid precipitation of IgM, it is important to add the ammonium sulphate slowly. An increased concentration of ammonium sulphate will cause more IgG to bind, which might be a problem if serum has been added to the cell culture medium. If there is IgG contamination of the purified IgM, the IgG can be removed by using HiTrap Protein A HP, HiTrap rProtein A FF, or HiTrap Protein G HP. Purification 1. Wash column sequentially with at least 5 column volumes of binding, elution and wash buffer.
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. Yield may be improved by using a slower flow rate or passing the sample through the column several times.
Low Molecular Weight Calibration Kit (LMW), reduced Lane 2. Crude periplasmic fraction, reduced Lane 3. Pool I, purified Protein A-(HisGly) 4His, reduced 1 2 3 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. The clone was a kind gift from Dr. C. Fuller and S. Brasher, Department of Biochemistry, University of Cambridge, UK. 4 Flow: Approx.