Activated Sepharose beads

Efficiencies less than 70% indicate that inefficient IgG immobilization has occurred. The cause of inefficient coupling is often the presence of a buffer component (free amines in Tris-HCl buffers, for example) that competes with primary amines of the antibody for binding at the active sites of the cyanogen bromide activated Sepharose beads. 

In another study, 5 -amino-C6 modified nucleic acid was immobilized onto cyanogen bromide activated Sepharose beads (Fig. 4B) [15]. The 100 pm-diameter beads were then subsequently transferred at the PDMS/air interface (Fig. 3D). 

Fig. 4 Nucleic acid immobilization. Immobilization of 5 -amino-C6 modified probes onto (a) carboxylate modified latex beads (1 im) via carbodiimide reaction and (b) Cyanogen bromide (CNBr) activated Sepharose beads (100 un). (0 cyclic structure, e.g. dicyclo-hexylcarbodiimide)...

A water-insoluble lactoperoxidase, linked to CNBr-activated Sepharose beads, capable of iodinating serum albumin, is commercially available (Worthington Biochemical Corp.), and may be useful where avoidance of contamination of the product by the enzyme is important. 

Figure 9. Direct immobilization in PDMS of beads bearing biologically active compounds. Stepl Sepharose beads bearing bioactive compounds are arrayed as 0.3 pi drop onto a flat PVC master Step 2 a PDMS solution is poured onto the spotted array Step 3 the PDMS-based array is peeled off from the master and ready to use.

Alterations in pH also can be responsible for the increase in solubility of loaded active agents. Glucose oxidase immobilized on sepharose beads were incorporated into ethyl vinyl acetate matrices along with insulin in the solid form. Glucose from blood enters these matrices, gets oxidized to glucuronic acid, and the decrease in pH increases the solubility of insulin, which diffuses out. The insulin in this case was modified by the addition of three extra lysine residues that ensured an isoelectric point of pH 7.4 for the molecule.33... 

Epichlorohydrin (l-chloro-2,3-epoxypropane). Widely available. Used to epoxy activate the Sepharose CL-6B beads or other surfaces. The extent of epoxy activation of beads may be determined see Note 2). A high-purity (+99%) or equivalent should be used. It is a very unstable compound and must be stored at 0-4°C in an anhydrous environment. Hazards Flammable, poison, toxic by inhalation or contact with skin, and if swallowed may cause cancer. Toxicity data LD50 90 mg/kg oral, rat. Should be handled in a fume hood with safety glasses and gloves, and treated as a possible cancer hazard. 

The CNBr-activated Sepharose 4B support (1 g dry weight) was swelled in 25 mL of hydrochloric acid (0.001 Af), and then washed with 100 mL of 0.5M NaCl, 0.1 M NaHC03 buffer at pH 8.3. To this support 5.5 mL of hydroxylapatite-purified heparinase (0.2 mg/mL protein with an activity of 88 units/mg protein in 0.2M phosphate buffer at pH 7.0) and 60 mg of heparin were added. The mixture was shaken overnight at 4°C, after which the beads were washed and blocked overnight at 4°C with a solution of lysine at pH 8.2 in 0.5M NaCl, 0.1M NaHC03 buffer solution. This support showed an uptake of 87% of the protein and an immobilization of 91% of the heparinase activity. 

Dissolve 1.1 g of CNBr in 2 ml of dimethylformamide. Add it slowly to the slurry over a period of 1-2 min. Then add 1.8 ml of 5 A/ NaOH dropwise over a period of 10 min followed by the addition of 100 ml of ice-cold H2O. Transfer the slurry to a sintered glass funnel and wash under slight suction with 3x 100 ml of ice-cold H2O followed by 100 ml of ice-cold 10 mM potassium phosphate buffer, pH 8.0. It is important that the beads are washed with gentle suction and are never allowed to be sucked dry onto the filter. After washing, transfer the beads in 4 ml of 10 mM potassium phosphate buffer (pH 8) in a 15-mi conical screwcapped tube and immediately add the ligated oligonucleotides. It is important to do the CNBr activation as rapidly as possible and to add the DNA immediately to the activated Sepharose, without any delay. 

Chemical immobilization procedures of bioluminescent enzymes such as firefly luciferase and bacterial luciferase-NAD(P)H FMN oxidoreductase to glass beads or rods [174, 175], sepharose particles [176], and cellophane films [177] have produced active immobilized enzymes. Picomole-femtomole amounts of ATP or NAD(P)H could be detected using immobilized firefly luciferase or bacterial luciferase-oxidoreductase, respectively. 

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