By GE HEalthcare
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Extra info for affinity chromatography principles and methods
Prepacked 5 ml column. HiTrap IgY Purification HP columns are packed with a thiophilic adsorption medium (2-mercaptopyridine coupled to Sepharose High Performance). The interaction between the protein and the ligand has been suggested to result from the combined electron donating- and accepting-action of the ligand in a mixed mode hydrophobic-hydrophilic interaction. Purification example Figure 20 shows the purification of a-Hb IgY from 45 ml of egg yolk extract (corresponding to one quarter of a yolk) and Figure 21 shows the SDS-PAGE analysis indicating a purity level of over 70%.
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.
Glutathione 10–12 mg recombinant GST/ml medium 450 cm/h* Sepharose 4 Fast Flow For packing high performance columns for use with purification systems and scaling up. Glutathione 8 mg horse liver GST/ml medium 75 cm/h* Sepharose 4B For packing small columns and other formats. * See Appendix 4 to convert linear flow (cm/h) to volumetric flow rate. d. of 5 cm. Purification examples Figure 22 shows a typical purification of GST fusion protein on GSTrap FF 1 ml with an SDS-PAGE analysis of the purified protein.
affinity chromatography principles and methods by GE HEalthcare