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5 M potassium sulphate. 8 M ammonium sulphate. Some monoclonal IgM may bind too tightly to the column for complete elution. The remaining IgM will be eluted during cleaning, but the high content of isopropanol will cause precipitation of IgM. Perform an immediate buffer exchange (see page 21) or dilute the sample to preserve the IgM. Lower concentrations of isopropanol may elute the IgM and decrease the risk of precipitation. Reuse of HiTrap lgM Purification HP depends on the nature of the sample and should only be considered when processing identical samples to avoid cross-contamination.

This group may be required for the specific interaction), then pre-activated media for ligand attachment via carboxyl, thiol or hydroxyl groups can be considered. These are described in the handbook, Affinity Chromatography: Principles and Methods from Amersham Biosciences. Pre-activated media are supplied in different prepacked column formats, in suspension or as dry medium. A pure ligand is required that has a proven reversible high affinity for the target molecule. Using an antigen or an anti-antibody as a ligand will give a high degree of purification.

High pH (up to pH 11). • Substances that reduce the polarity of a buffer may facilitate elution without affecting protein activity: dioxane (up to 10%), ethylene glycol (up to 50%). NHS-activated Sepharose is the first choice for the preparation of immunospecific media. CNBr-activated Sepharose media offer a well-established option for the attachment of larger ligands and can be an alternative to NHS-activated Sepharose. 49 50 Chapter 4 Immunoprecipitation Immunoprecipitation is a highly specific technique for the analysis of target antigens from crude cell lysates.

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