By Meir Wilchek, Irwin Chaiken (auth.), Pascal Bailon, George K. Ehrlich, Wen-Jian Fung, Wolfgang Berthold (eds.)
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Additional resources for Affinity Chromatography: Methods and Protocols
This concentration will give a stable crosslinking bond to the Protein G that will not leach an antibody with low pH elution buffers. However, in cases where it is necessary to achieve the highest possible antigen capacity, it has been demonstrated that near theoretical maximal capacity could be achieved by determining the critical minimum (and therefore optimum) concentration of DMP. This suggests that too high a concentration may result in intrachain crosslinks that may be in close proximity to the antigen binding site.
4 kDa). Staining of the protein bands was achieved with Zoion Fast Stain Concentrate (Newton, MA) using the manufacturer’s instructions. Results can be seen in Fig. 3. Analyzing the proteins shows that high purity can be achieved in a single step. Purity is equivalent to proteins prepared using conventional packed-bed column chromatography with clarified cell culture supernatants and extracts. 9. 1. HAT The activity of HAT is determined by a competitive IL-2 binding assay in which IL-2 competes against HAT for binding to the IL-2R (21).
Ties. Ligand denaturation does not constitute a problem, such as in the case of protein A, and TG19318 columns can withstand a large array of harsh sanitizing agents with no capacity losses. In addition, the low toxicity of TG19318 and the low molecular weight of the resulting fragments reduces considerably the problems of contamination by leaked ligand, as is the case for protein A. Preliminary experiments suggest that the ligand is more stable to proteolytic digestion when coupled to solid supports, and the enzymatic activity normally found in crude feedstock derived from cell culture supernatants does not lead to noticeable loss of capacity.