Agarose aminated

At high pH (10), Traut s reagent also is reactive with aliphatic and aromatic hydroxyl groups, although the rate of reaction with these groups is only about 0.01 that of primary amines. In the absence of amines, however, carbohydrates such as agarose or cellulose membranes can be modified to contain sulfhydryl residues (Alagon and... 

Ninhydrin (1,2,3-triketohydrindene monohydrate). Widely available chemical, light-sensitive. Ninhydrin reacts with free amines (2 1 molar ratio), giving a purple product (Ruhemann s purple resonance structure). Used for a qualitative test to determine the presence of aliphatic amines on the agarose beads as a 0.2% w/v solution in ethanol (see Note 4). Hazards Harmful if swallowed skin, eye, and respiratory irritant. Toxicity data LD50 78 mg/kg intraperitoneal, mouse. Should be handled in a fume hood with safety glasses and gloves. 

Amines. The amines used to sequentially substitute the chlorines of cyanuric chloride free or immobilized onto agarose vary from case to case. In general, the amines should be dissolved in an appropriate buffer, either an aqueous solution or an organic solvent such as dimethylformamide. Take precautions related to the re-ac-tivity of each compound, which in some cases may involve the protection of... 

Amination of agarose beads. The washed epoxy-activated gel is suspended in 1 mL of distilled water/g of gel in a 1-L conical flask. About 1.5 mL of ammonia/ g of gel are added and the gel incubated for 12 h at 30°C in a rotary shaker. Alternatively, 1,6-diaminohexanediamine (5 eq.) can be used to aminate the gel, yielding resins with a six-carbon spacer arm. The aminated gel is washed with 40 mL of distilled water/g of gel on a sinter funnel, and stored in 20% v/v ethanol at 0-4°C. The extent of amination is determined as described in Note 3. Aminated beads can also be purchased from Amersham Biosciences. 

Each immobilized ligand matrix (1.5 pL) is placed on a microscope slide and observed under a fluorescence microscope (FITC-/iexc = 495 nm A,em = 525 nm). The control experiments consist of repeating the procedure described above using Sepharose CL-6B, aminated agarose, and control ligand 0/0. 

Q-Sepharose 6% Cross-linked agarose T rimethylamino (quaternary amine) 90 (45-165)... 

In the second case, an immobilization chemistry is required to graft avidin onto the beads. When using agarose beads, the microspheres were purchased as terminated with aldehyde groups to which proteins could be linked via reductive amination (Fig. 2B) [6]. Another possibility is to use commercially available neutravidin coated beads [7]. This affinity immobilization system enables the achievement of perfectly orientated DNA probes since the bi-... 

The enzyme preparation used was purified to apparent homogeneity from human liver by affinity chromatography on agarose-e-aminocaproylfucos-amine resin. 

The most effective supports were those prepared by the attachment of 17/3-oestradiol 17-hemisuccinate to agarose derivatives containing albumin or the poly-L-lysine or poly-DL-amine spacers. Non-specific adsorption of non-receptor proteins was reported to be minimal and the described procedures gave purification between 10000- and 100000-fold with 30 to 50% yields in a single step. 

Van Leemputten E and Horisberger M. Immobilization of enzymes on magnetic particles. Biotechnol. Bioeng. 1974 16 385-396. lost R, Miron T, and Wilchek M. The mode of adsorption of proteins to aliphatic and aromatic amines coupled to cyanogen bromide-activated agarose. Biochim. Biophys. Acta 1974 362 75-82. 

Traditionally in biochemistry (e.g. for affinity chromatography), enzymes have been covalently linked to carbohydrates such as agarose and dextran (Sepharose, Sephadex). The most widely used activation method is the cyanogen bromide (CNBr) method, yielding isourea and imidocarbonate functionalities that react with amines on the enzyme to produce N-substituted carbamate linkages. 

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