Affinity purification of antibodies by using Ni2+-resins on which inclusion body-forming proteins are immobilized |
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Authors: | Chumpia Worawan Ohsato Takashi Kuma Hiroyuki Ikeda Shogo Hamasaki Naotaka Kang Dongchon |
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Institution: | Department of Clinical Chemistry and Laboratory Medicine, Kyushu University Graduate School of Medical Sciences, Fukuoka 812-8582, Japan. |
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Abstract: | Bacterially expressed recombinant proteins are widely used for producing specific antibodies. Unfortunately, many recombinant proteins are recovered as insoluble materials, so-called inclusion bodies. Inclusion bodies are rather advantageous from a point of view of immunogens because fairly pure proteins can be feasibly extracted from the inclusion bodies. However, we encounter a problem with an insoluble protein when we make an antigen-immobilized column for affinity purification of antibodies because we need a soluble protein in usual immobilization methods. Histidine-tagged proteins can be bound to Ni(2+)-resins in buffer containing 6M guanidine-HCl, in which most insoluble proteins are solubilized. Taking advantage of this feature, we have successfully purified antigen-specific antibodies by directly using Ni(2+)-resins onto which denatured proteins are bound. |
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