By Amersham Biosciences
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Additional info for Affinity Chromatography
45 Cleaning These procedures are applicable to Glutathione Sepharose 4 Fast Flow and Glutathione Sepharose 4B. 1. 5) buffers. 2. Repeat the cycle 3 times. 3. Re-equilibrate immediately with 3–5 column volumes of binding buffer. If the medium is losing binding capacity, this may be due to an accumulation of precipitated, denatured or non-specifically bound proteins. To remove precipitated or denatured substances: 1. Wash with 2 column volumes of 6 M guanidine hydrochloride. 2. Wash immediately with 5 column volumes of binding buffer.
One molecule of protein A can bind at least two molecules of IgG. Both protein A and a recombinant protein A are available from Amersham Biosciences. These molecules share similar specificities for the Fc region of IgG, but the recombinant protein A has been engineered to include a C-terminal cysteine that enables a single-point coupling to Sepharose. Single point coupling often results in an enhanced binding capacity. The binding strength of protein A for IgG depends on the source species of the immunoglobulin as well as the subclass of IgG (see Table 2).
Wash the column with at least 5 column volumes of binding, elution and wash buffer. 2. Equilibrate with 5 column volumes of binding buffer. 3. Apply the sample. 4. Wash with at least 10 column volumes of binding buffer or until no material appears in the eluent, as monitored at A280. 5. Elute the IgY with 10 column volumes of elution buffer. 6. Wash the column with 8 column volumes of wash buffer. 7. Immediately re-equilibrate the column with 5 column volumes of binding buffer. 8 M Na2SO4. The sample should have the same concentration of Na2SO4 as the binding buffer.