Sephrose 4 Fast Flow matrix.docVIP

Sepharose 4 Fast Flow matrix Sepharose 4 Fast Flow is a highly cross-linked agarose matrix. In its preactivated CNBr form, it offers much improved performance when compared with the well established CNBr-activated Sepharose 4B. The Sepharose 4 Fast Flow matrix has higher rigidity and can thus be run at high flow rates (see Table 1). The higher mechanical strength of the cross-linked matrix makes it wellsuited for use in large columns. Scaling up a purification developed on CNBractivated Sepharose 4 Fast Flow is therefore simple and more predictable. The coupled product is stable at low pH, which is often required for elution from some immunoadsorbents. For applications that require operation at high pH, note that the amide bond formed when using the companion product NHS-activated Sepharose 4 Fast Flow is stable up to pH 13 for normal use. 2. Coupling CNBr-activated Sepharose 4 Fast Flow is supplied freeze-dried in the presence of additives. These additives need to be washed away at low pH (pH-2–3) before coupling the desired ligand. The use of low pH (pH2–3) preserves the activity of the reactive groups, which otherwise hydrolyse at high pH. In order to retain maximum binding capacity of CNBr-activated Sepharose 4 Fast Flow prior to coupling the ligand, use cold (0–4 °C) solutions. The time interval between washing and coupling must be minimised; therefore preparations of all required solutions prior to coupling is recommended. 1. Prepare the coupling solution, i.e. dissolve the ligand to be coupled in a suitable coupling buffer, e.g. 0.1 M NaHCO3 pH 8.3 containing 0.5 M NaCl. For good coupling efficiency avoid unnecessarily dilute solutions (Recommended ratio of volumes, coupling solution/medium is 0.5:1). The coupling pH depends on the ligand. Normally pH in the range 7–9 is used. 2. CNBr-activated Sepharose 4 Fast Flow is supplied freeze-dried with sugar additives and is washed initially with 10–15 medium volumes of cold 1 mM HCl, see Fig 1. Use small wash port