Purification of sheep immunoglobin G using protein A trapped in sol-gel glass
✍ Scribed by Rivka Zusman; David A. Beckman; Igor Zusman; Robert L. Brent
- Publisher
- Elsevier Science
- Year
- 1992
- Tongue
- English
- Weight
- 825 KB
- Volume
- 201
- Category
- Article
- ISSN
- 0003-2697
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✦ Synopsis
An effective method for purifying immunoglobulins has been developed utilizing a sol-gel glass support system. Sol-gel glass is an effective support for chemically active ligands entrapped in this medium at room temperature. There are two problems associated with the utilization of such sol-gel glasses for entrapping macromolecules. One is the phenomenon of nonspecific absorption of proteins onto the glass. The second is that only a portion of the entrapped molecules may retain their biological activity. In the present study a sol-gel glass was treated with gamma-aminopropyltriethoxysilane to provide a matrix that eliminated nonspecific absorption of proteins. The method of entrapping molecules was modified to increase the proportion of entrapped molecules that retained their reactivity. Protein A was entrapped in the modified sol-gel glass column and used to purify IgG from sheep sera by affinity chromatography. The purity of the IgG, as determined by SDS-PAGE, was comparable to that obtained from commercially available protein A columns and, if the capacity of the column was not exceeded, the yield approached 100%. Although the quality and quantity of the yields were comparable, the methodology described herein can be accomplished more rapidly and with greater ease. Furthermore, the sol-gel glass ligand preparation is extremely stable and can be reused for isolation of gamma-globulin. The technique has great potential for isolating macromolecules utilizing various ligands.
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