Solid supports sepharose

The interfering effect of non-specific hydrophobic interactions with the spacer molecule is avoided if hydrophilic spacers are used [28,49]. The use of hydrophilic spacers also prevents another undesirable situation that may occur when a hydro-phobic affinant is coupled to a long, flexible hydrophobic chain. When Holguin and Cardinaud [50] used Sepharose-iV -p-amino-M-hexyladenine, they assumed that under the effect of the flexible chain the active part of the affinity ligand came into close proximity of the solid support and that therefore an effective interaction with the transfer enzyme could not take place. 

Sepharose and LH-20 [165-167] have been used as supports for SPS. Both are synthetic dextran polymer beads composed of fibers in a porous network. The fragile nature and acid lability of the glycosidic bonds of the dextran are serious limitations to the use of these materials as solid supports. However, they are perfectly compatible with aqueous buffers and can be used in combined chemoenzymatic syntheses [167]. 

Marine bacterial luciferase can be immobilized on to a variety of solid supports (e.g., Sepharose, nylon) and it has also been coimmobilized with NAD(P)H FMN oxidoreductase. The close proximity of the two enzymes leads to efficient channeling of FMNH2 produced by the oxidoreductase to the luciferase. More extensive coupled coimmobilized systems have been prepared in which all of the enzymes in the enzymatic conversion of glucose to alcohol were coimmobilized with bacterial luciferase and the oxidoreductase, and the bioluminescent enzymes were used to monitor different stages in the biotransformation of glucose. 

Antibodies are affinity purified by binding them to immobilized antigen. The most common solid support for immobilizing antigen is cyanogen bromide-activated Sepharose. Almost all antigens containing amino... 

In order to immunoprecipate antigens, the revelant antibody is preferably coupled to the solid support (protein A-sepharose beads), but alternatives are mentioned in Note 5. 

Actually, Harbury s approach exploits DNA templating in a different way. Strictly speaking, the library is not synthesized by templated reactions. DNA templates are not used to bring reactants in proximity to boost the effective molarity instead DNA templating is the mean to spatially partition the templates into sub-pools. The advantage of this approach is mostly that chemical building blocks can be directly used without the need to couple to a DNA strand and no cleavable linkers are needed second, the chemical reactions take place on solid support (DEAE Sepharose column), therefore potentially more chemical reactions can be applied to create libraries with more diverse structures. However, so far there has been no report of library synthesis other than peptidic structures. 

Because of the complexity of the enzymatic systems involved in coenzyme Bi2 chemistry there are several reports on the purification of B 12-dependent enzymes or B 12-binding proteins by vitamin B12 affinity adsorbents. In fact, for purification of enzymes or proteins, affinity chomatography has been widely used as one of the most attractive methods (270). For that purpose, the synthesis of a cobalamin-Sepharose insoluble support has been prepared and applied to the purification of iV -methyltetrahydrofolate-homocysteine cobalamin methyltransferase from E. coll The scheme for the synthesis of the solid support is summarized in Fig. 6.14. 

Finally, attention was turned to neutral steroids, and as a representative model, cholic acid methyl ester 17 was chosen. The initial attempt to oxidize 17 in a biphasic aqueous buffer-ethyl acetate system, by the action of the usual NAD-dependent 7a-HSDH, was unsuccessful. To overcome this problem, a new 7a-HSDH, namely, the NADP-dependent 7a-HSDH from C. absonum [16], was considered. Unfortunately, the subsequent reduction of 65 occurred with a low degree of conversion (less than 20%), probably due to enzyme inactivation. The stability of 7p-HSDH was not improved by immobilization on solid supports such as Eupergit C or activated Sepharose [6]. 

Selected signature libraries may be immobilized on a solid matrix such as activated silica resin, cellulose microporous modified membranes [66], Sepharose , magnetic beads based on MagaPhase technology. The affinity support obtained is used for IgM antibodies parting. 

Cibacron Blue is a blue, polyaromatic, sulfonated dye (Fig. 6). It can be attached, as an affinity li nd, to solid matrix supjmrts (e.g. dextran, agarose) by the reaction of the triazine ring with free hydroxyl groups of the supports. The conditions of this triazine coupling method have been described by Bohme Such a dye affinity sorbent is also produced commercially, e.g. under trade name Blue Sepharose CL-6B (Cibacron Blue F3G-A covalently bound to the cross-linked agarose gel Sepharose CL-6B ) by Pharmacia, Sweden. 

To affinity purify the subtracted serum, an appropriate affinity support must be selected. The antigen is immobilized on a solid phase support. For example, synthetic peptides or proteins can be attached to CNBr Sepharose (Pharmacia) or Affigel 15 (Bio-Rad), which couple through primary amines. If use of an amine is not optimal, one may couple via carbodiimide bonding utilizing EAH Sepharose 4B (Pharmacia) crosslinked with EDC (Pierce) In general, 20 mg of peptide per mL of gel should be sufficient. 

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