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New PDF release: Affinity Chromatography

By Amersham Biosciences

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0 1 ml min SDS-PAGE on ExcelGel™ SDS Gradient 8–18% using Multiphor™ II followed by silver staining. Lane 1. Low Molecular Weight (LMW) Calibration kit, reduced, Amersham Pharmacia Biotech Lane 2. Cytoplasmic extract of E. coli expressing GST fusion protein, 1 g cell paste/10 ml Lane 3. GST fusion protein eluted from GSTrap FF 1 ml Fig. 22. Purification of a GST fusion protein. 43 Figure 23 shows how the purification of a GST fusion protein can be scaled up 20-fold from a GSTrap FF 1 ml column to a GSTPrep FF 16/10 column.

Reuse of HiTrap IgY Purification HP depends on the nature of the sample. To prevent cross-contamination, columns should only be reused with identical samples. Media characteristics HiTrap IgY Purification HP Ligand and density pH stability* 2-mercaptopyridine 3 mg/ml Long term 3–11 Short term 2–13 Mean particle size 34 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.

0 Centrifuge samples (10 000 g for 10 minutes) to remove cells and debris. 45 µm filter. If needed, adjust sample conditions to the pH and ionic strength of the binding buffer either by buffer exchange on a desalting column (see page 133) or by dilution and pH adjustment. A HiTrap column can be used with a syringe, a peristaltic pump or connected to a liquid chromatography system, such as ÄKTAprime. 1. Equilibrate the column with 5 column volumes of binding buffer. 2. Apply sample. 3. Wash with 5–10 column volumes of the binding buffer to remove impurities and unbound material.

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