By Amersham Biosciences
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Extra info for Antibody Purification
Table 15 presents recommendations for the products that would be most useful for laboratory scale purification. Affinity chromatography is used to achieve a high resolution purification in the first capture step. Gel filtration is used to separate dimers from monomers and remove any leached ligand in the polishing step. Step Increasing scale Capture Specific ligand bound to HiTrap NHS-activated HP, 1 ml or 5 ml Specific ligand bound to NHS-activated Sepharose Fast Flow Polishing (select medium according to size of target molecule and contaminants) Superdex 200 10/300 GL (Tricorn™) Superdex 75 10/300 GL (Tricorn) HiLoad 16/60 Superdex 200 pg HiLoad 26/60 Superdex 200 pg HiLoad 16/60 Superdex 75 pg HiLoad 26/60 Superdex 75 pg HiLoad 16/60 Superdex 30 pg HiLoad 26/60 Superdex 30 pg Buffer exchange/desalting HiTrap Desalting HiPrep 26/10 Desalting Table 15.
Hydrophobicity IgG is more hydrophobic than many other proteins and so precipitates more readily in ammonium sulphate. Solubility IgG very soluble in aqueous buffers. Lowest solubility (specific to each antibody) near pI or in very low salt concentration. Temperature stability Relatively stable at room temperature (but specific to each antibody). pH stability Often stable over a wide pH interval, but unstable in very acidic buffers (specific to each antibody). Carbohydrate content 2–3% for IgG, higher for IgM (12%), most carbohydrate is associated with Fc region of the heavy chains.
General instructions for use are given in Appendix 3. To increase capacity HiTrap columns can be linked in series. Most media are available for packing larger columns. Always check availability if the intention is to scale up. Custom-designed affinity media can be produced. Reuse of affinity media depends on the nature of the sample and should only be considered when processing identical samples to avoid cross-contamination. Step Increasing scale Capture HiTrap Protein G HP, 1 ml or 5 ml HiTrap Protein A HP, 1 ml or 5 ml HiTrap rProtein A FF, 1 ml or 5 ml Protein G Sepharose Fast Flow Protein A Sepharose Fast Flow rProtein A Sepharose Fast Flow MabSelect™ Polishing (select medium according to size of target molecule and contaminants) Superdex 200 HR 10/30 Superdex 75 HR10/30 HiLoad™ 16/60 Superdex 200 pg HiLoad 26/60 Superdex 200 pg HiLoad 16/60 Superdex 75 pg HiLoad 26/60 Superdex 75 pg HiLoad 16/60 Superdex 30 pg HiLoad 26/60 Superdex 30 pg Buffer exchange/desalting HiTrap Desalting 5 ml HiPrep 26/10 Desalting Table 12.