By Meir Wilchek, Irwin Chaiken (auth.), Pascal Bailon, George K. Ehrlich, Wen-Jian Fung, Wolfgang Berthold (eds.)
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Extra resources for Affinity Chromatography: Methods and Protocols
4. Stop the crosslinking reaction by exchanging with 5 vols of blocking buffer. ) After the first wash, incubate the gel with a second wash for 5 min before centrifuging. Repeat washing twice. 5. Incubate beads for 5 min in 2 vol of elution buffer. This wash will remove any IgG that has not been covalently bound to the gel. 6. 02% azide for storage. 3. Confirm the Stability of the Antibody-Crosslinked Protein G Sepharose 1. Prepare a portion of the antibody-bound Protein G Sepharose before and after crosslinking into a 50% slurry with PBS.
50, 1495–1502. 16. , Kaffka, K. , and Killian, P. L. (1987) Structure-function analysis of human interleukin-2: Identification of amino acid residues required for biological activity. J. Biol. Chem. 262, 5723–5731. 17. Kreitman, R. , Batra, J. , Chaudhary, V. , Fitzgerald, D. , and Pastan, I. (1993) Single-chain immunotoxin fusions between anti-Tac and Pseudomonas exotoxin: Relative importance of the two toxin disulfide bonds. Bioconjugate Chem. 4, 112–120. 18. , Anderson, L. , Podlaski, F. , Jenson, J.
Recent works pointed out the possibility of using alternative ligands for the affinity purification of IgM. Immobilization of mannanbinding protein (MBP) on solid supports led to affinity media useful for IgM isolation based on a temperature-dependent interaction of the ligand with the immunoglobulins (2). The use of immobilized MBP for the purification of IgM is based on the adsorption in the presence of calcium at a temperature of 4°C, and the room temperature-dependent elution of adsorbed immunoglobulins in the presence of ethylenediaminotetraacetic acid (EDTA).
Affinity Chromatography: Methods and Protocols by Meir Wilchek, Irwin Chaiken (auth.), Pascal Bailon, George K. Ehrlich, Wen-Jian Fung, Wolfgang Berthold (eds.)