| Triphenylmethyl Sepharose (trityl Sepharose), a hydrophobic resin with varying degree of tritylation is useful for multiple purposes. 5'-tritylated oligonucleotides binding hydrophobically to low trityl Sepharose (20 mM trityl) retain their hydrogen bonding specificities for complementary sequences. The salt, dielectric constant and temperature dependance of these non-covalently anchored ligands permits the isolation of complementary RNA's including fibroin mRNA. Similarly, tritylated biospecific affinity ligands for proteins binding to low trityl Sepharose permits the isolation of their biospecific protein, such as pancreatic ribonuclease. This constitutes a novel mode of attaching affinity ligands to solid supports, is more convenient than existing methods, and proceeds with 100% yield.;The ease of immobilization, enhanced stability, quick and complete removal of products and reusability makes these a valuable contribution to molecular biology techniques.;Medium trityl Sepharose (20-40 mM trityl) has a high binding specificity for poly(A) and poly(A)containing RNA, equivalent to the conventional dT-cellulose. Poly(A)containing RNA was isolated from rabbit liver, WI38 cells, HeLa cells and polio virus infected HeLa cells using dT-cellulose and medium trityl Sepharose. Nearly all proteins/enzymes checked bind to these columns and retain good activity from several weeks to several months. Nucleases, polymerases, polynucleotide kinase, proteases and restriction endonucleases have been immobilized hydrophobically. Nearly quantitative immobilization of enzymes compares very favourably with current covalent attachment methods. Hydrophobic immobilization of enzymes probably mimic the in vivo membrane associated nature of most enzymes/proteins. |
