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Additional resources for affinity chromatography principles and methods
Purification example Figure 29 shows automatic on-line monitoring of the production of a secreted fusion protein during fermentation. The fusion protein, ZZ-IGF-1 is insulin-like growth factor 1 fused with a derivative of protein A (designated ZZ), expressed in E. coli. 0 Time (min) A 600 nm 120 Conc. (mg/ml) 600 Conc. A600 100 500 80 400 60 300 40 200 20 100 0 10 20 30 40 0 50 Time (h) Fig. 29. A) Chromatogram of a sample taken at one time point during fermentation. B) Results from automatic monitoring of the product concentration during fermentation.
Wash column sequentially with at least 5 column volumes of binding, elution and wash buffer. 2. Equilibrate column with 5 column volumes of binding buffer. 3. Apply the sample. 4. Wash out unbound sample with 15 column volumes of binding buffer or until no material appears in the eluent (monitored at A280). 5. Elute the IgM with 12 column volumes of elution buffer. 6. Wash the column with 7 column volumes of wash buffer. 7. Immediately re-equilibrate the column with 5 column volumes of binding buffer.
Enhanced binding capacity. Prepacked columns. rProtein A Human IgG, > 50 mg/ml medium 300 cm/h** Sepharose 4 Mouse IgG, 8–20 mg/ml medium Fast Flow* Enhanced binding capacity. Supplied as a suspension ready for column packing. MabSelect™ Human IgG, approx. 30 mg/ml medium 500 cm/h** (recombinant protein A ligand) For fast processing of large sample volumes. Retains high binding capacity at high flow rates. Supplied as a suspension ready for column packing. * Protein A Sepharose 4 Fast Flow and rProtein A Sepharose Fast Flow have a higher binding capacity, a more rigid matrix and provide more convenient alternatives to Protein A Sepharose CL-4B, which must be rehydrated before column packing.