PHARMACIA FINE CHEMICALS's Affinity Chromatography: Principles and Methods PDF

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1. 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.

As an affinity ligand, protein A is coupled to Sepharose so that these regions are free to bind IgG. One molecule of protein A can bind at least two molecules of IgG. Both protein A and a recombinant protein A are available from Amersham Biosciences. These molecules share similar specificities for the Fc region of IgG, but the recombinant protein A has been engineered to include a C-terminal cysteine that enables a single-point coupling to Sepharose. Single point coupling often results in an enhanced binding capacity.

HiTrap Benzamidine FF (high sub) provides a simple, ready to use solution for this process. Figure 30 shows the partial structure of Benzamidine Sepharose 4 Fast Flow (high sub) and Table 4 gives examples of different serine proteases. OH OH Sepharose O O O H N NH N NH 2 H Fig. 30. Partial structure of Benzamidine Sepharose 4 Fast Flow (high sub). Table 4. Examples of different serine proteases. 0 (saliva) Purification options Binding capacity Maximum operating flow Comments HiTrap Benzamidine FF (high sub) Trypsin, > 35 mg/column Trypsin, > 175 mg/column 4 ml/min (1 ml column) 15 ml/min (5 ml column) Prepacked columns**.

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Affinity Chromatography: Principles and Methods by PHARMACIA FINE CHEMICALS


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