The instructions state that my species of albumin binds, but it is coming through in the wash. What could be wrong?
You might be overloading the sample. Apply a smaller sample to the support. Each disc can bind at least 2 mg of human albumin. Alternatively, the buffer salt concentration might not be optimal. Prepare a buffer with less salt (e.g., 25-50 mM NaCl instead of 75 nM). Will this bind (resulting in the loss of) IgG and other proteins besides albumin? Yes. Some optimization and validation with respect to other proteins of interest may have to be performed. The immobilized Cibacron Blue can bind enzymes that require adenylic cofactors, such as NAD+, NADP+ and ATP. Also transferases, polymerases, nucleases, hydrolases, synthetases, and other CoA-dependent enzymes can be purified using this as a general purification system.
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