affinity chromatography principles and methods by GE HEalthcare

By GE HEalthcare

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Long term 3–9 Short term 2–10 90 µm * Long term refers to the pH interval over which the medium is stable over a long period of time without adverse effects on its subsequent chromatographic performance. Short term refers to the pH interval for regeneration, cleaning-in-place and sanitization procedures. Chemical stability Stable in all common aqueous buffers. Storage Wash media and columns with 20% ethanol (use approximately 5 column volumes for packed media) and store at +4 to +8 °C. HiTrap Protein A HP, Protein A Sepharose 4 Fast Flow, HiTrap rProtein A FF, rProtein A Sepharose 4 Fast Flow, MabSelect Protein A is derived from a strain of Staphylococcus aureus and contains five regions that bind to the Fc region of IgG.

GE Healthcare offers a recombinant form of protein G from which the albumin-binding region of the native molecule has been deleted genetically, thereby avoiding undesirable reactions with albumin. 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.

Coli expressing GST-DemA, 1 g cell paste/5 ml Flow-through from GSTrap FF 5 ml GST-DemA eluted from GSTrap FF 5 ml Extract of E. 0 A B C Fig. 24. Using GSTrap FF with a syringe. A: Prepare buffers and sample. Remove the column’s top cap and twist off the end. B: Equilibrate column, load the sample and begin collecting fractions. C: Wash and elute, continuing to collect fractions. 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.

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