Activity gel

Asphaltenes seem to be relatively constant in composition in residual asphalts, despite the source, as deterrnined by elemental analysis (6). Deterrnination of asphaltenes is relatively standard, and the fractions are termed / -pentane, / -hexane, / -heptane, or naphtha-insoluble, depending upon the precipitant used (5,6,49). After the asphaltenes are removed, resinous fractions are removed from the maltenes-petrolenes usually by adsorption on activated gels or clays. Recovery of the resin fraction by desorbtion is usually nearly quantitative. 

Use the activated gel immediately for coupling (e.g.. Protocol 3.6.2), because the reactive groups hydrolyze fast. 

Ten grams of Sepharose are sucked off on a sintered-glass funnel. First wash with 3 vol. water, then with 3 vol. 30% acetone in water (v/v), and then with 3 vol. 60% acetone (v/v) in water. Suspend the gel in 10 ml of 60% acetone and cool to 0 °C. Add the needed amount CDAP dropwise (cf. Table 3.9) followed by the respective amount of triethylamine (TEA) to the stirred gel suspension. Suck off the gel after 2 min and wash with a tenfold volume of Soln. D. The activated gel is stable for 1 h. 

Weigh in 5 mg of sucked-off activated gel into a test tube and add 200 pi Soln. A. Close the tube and mix for 15 min at RT. Add 2.80 ml ddH20 and allow to setde or centrifuge. Read the supernatant at 588 run in a photometer. If necessary, dilute with water. 

Wash freshly activated gel (see Protocol 3.6.1) with huge amounts of ice-cold water or in the case of commercially available activated gels, e.g., BrCN-activated Sepharose 6B, with ice-cold 0.001 N HCl. 

The immobilization of any other protein to cyanogen bromide-activated or N-hydroxysuccinimide- (NHS) activated gels is done the same way. 

Epoxy-activated gels are especially used for immobilization of carbohydrates. 

Mix 30 g of wet Sepharose CL-4B with 40 ml of Soln. A, 5 ml epichiorohydrin and 25 ml pure water. Incubate at 40 °C with gentle agitation for 2 h. Then wash on a sintered-glass funnel with 500 ml water and suck off remaining water. Activated gel is stable at 4 °C for several days. 

A wide variety of activated gels is now commercially available. The most widely used are described as follows ... 

Wash the activated gel thoroughly with water until the filtrate is no longer acidic (see Note 23). 

Wash activated gel with 200 mL of coupling solution (0.1M sodium carbonate, pH 9 0) using vacuum filtration, and resuspend in 75 mL of coupling solution... 

The activated gel can be stored by washing thoroughly in acetone and kept as a suspension in acetone at 4°C. 

Extensively wash the activated gel with distilled water on a supporting filter. The washed gel can be stored in water at 4°C, for up to 10 d... 

Attachment of specific ligands to activated gels R = ligand... 

Fia. 14. Polyacrylamide gel electrophoresis at various purification steps. Gels 1 to 4 were stained for acid phosphatase activity gel 5 was stained for protein. A current of 4 mA/gel was applied to gels 1-3 for 2 hr and to gels 4 and 5 for 6 hr. Gel 1, homogenate 2, DEAE-cellulose peak II 3, DEAE-cellulose peak I 4 and 5, crystalline enzyme. From Igarashi and Hollander (4). 

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. 

Nucleophilic substitution of R. Cyanuric chloride activated gel is divided into n aliquots, where n is the number of different amines used to synthesize the combinatorial library. A twofold molar excess (relative to the amount of amination of the gel) of each amine is dissolved in the appropriate solvent (1 mL/g gel) (see Note 8). The n aliquots are suspended in the previous mixture, and incubated at 30°C in a rotary shaker (200 rpm) for 24 h. After this period, each R,-substituted gel is thoroughly washed on a sintered funnel with the appropriate buffer for each amine. The resulting gel is stored in 20% v/v ethanol at 0-4°C or used immediately for R2 substitution (Fig. 3). 

Extent of epoxyactivation of agarose beads. Sodium thiosulphate (1.3 M, 3 mL) is added to 1 g of epoxy-activated gel and incubated at room temperature for 20 min. This mixture is neutralized with 0.1 MHC1 and the amount of HO used is recorded. The volume of 0.1 MHO added corresponds to the number of OH moles released (10 pmol for each 100 pL added), which equals the pmol of epoxy groups/g gel. Therefore, the extent of epoxy activation is expressed as pL HC1 used/10 pmol/g gel. The protocol usually results in 25 pmol epoxy groups/g moist weight gel. 

Table 5. /. The properties of initial and activated gels of titanium and zirconium dioxides [12].

The strengthening of bonds in the monoclinic modification causes an increase of the crystallization temperature of activated gels. The activation of TiOj gel causes anatase formation and simplifies further transformation into rutile (T, decreases). 

To prepare the silica gel (Anasorb, 130-140 mesh, Analabs, Inc.) for use, a portion is placed in an evaporating dish and baked overnight at 275° in a clean oven (15 g, for example, will fill five columns). Longer heating is permissible. When removed from the oven, the gel is immediately poured into a small bottle which is then tightly capped and allowed to cool to room temperature. Activated gel is not allowed to stand more than a day prior to use. Unused gel can be reheated. Hydrocarbon-free nitrogen is tested as part of column blank measurement. 

M sodium borate, 1 mM EDTA, 100 mM DTT (3 column volumes), followed by PBS + 2 mM EDTA (5 column volumes) and connected to the second column filled with the thiopyridine-activated gel. The dissolved precipitate is applied with a flow of 0.1 ml/min, and the column set is washed with column buffer, about 10 column volumes overnight. The reducing column is disconnected and the second column is eluted with the latter buffer supplemented with 10 mM DTT, at a flow of 0.1 ml/min. Fractions of about 1/5 column volume are collected and assayed by SDS-PAGE and the hemolytic activity determined. 

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