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5. 5 with 30% isopropanol. 41 The sample must have the same concentration of ammonium sulphate as the binding buffer. 8 M. Stir slowly and continuously. 45 µm filter immediately before applying it to the column. 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.

Recombinant protein G contains two Fc binding regions. Protein G Sepharose is a better choice for general purpose capture of antibodies since it binds a broader range of IgG from eukaryotic species and binds more classes of IgG. Usually protein G has a greater affinity than protein A for IgG and exhibits minimal binding to albumin, resulting in cleaner preparations and greater yields. The binding strength of protein G for IgG depends on the source species and subclass of the immunoglobulin. The dynamic binding capacity depends on the binding strength and also on several other factors, such as flow rate during sample application.

Table 14 gives examples of some typical binding and elution conditions that have been used with Protein A Sepharose. 0 3–4 (using thiocyanate) Usually elutes by pH 3 Mouse Rat Table 14. Binding strengths are tested with free protein A and can be used as guidelines to predict the binding behaviour to a protein A purification medium. However, when coupled to an affinity matrix the interaction may be altered. For example, rat IgG1 does not bind to protein A, but does bind to Protein A Sepharose. With some antibodies, such as mouse IgG1, it might be necessary to add sodium chloride up to a concentration of 3 M in the binding buffer to achieve efficient binding when using protein A.

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