Crosslinking phosphate

Calcium, like oxygen, was useful for life s fortifications—but, also like oxygen, it would have been toxic at first. This is reflected in one of the major chemical balances for life from Chapter 5 eject calcium, import magnesium. When the calcium first saturated ocean waters, life kept shoveling it out like so much snow and bid it good riddance lest it crosslink phosphate-rich DNA. 

A similar mechanism was postulated for the Ca " -dependent inactivation of Ca -ATPase by ATP-imidazolidate [380] that results in intramolecular crosslinking with the formation of a new protein band of 125 kDa. In both cases the reactive carboxyl group was suggested to be the phosphate acceptor Asp351. 

Anionic Association Polymer. Another type of lost circulation agent is a combination of an organic phosphate ester and an aluminum compound, for example, aluminum isopropoxide. The action of this system as a fluid loss agent seems to be that the alkyl phosphate ester becomes crosslinked by the aluminum compound to form an anionic association polymer, which serves as the gelling agent [1488]. 

A similar process allows reacting triethyl phosphate and phosphorous pentoxide to form a polyphosphate in an organic solvent [871]. An excess of 1.3 moles of triethyl phosphate with respect to phosphorous pentoxide is the most preferred ratio. In the second stage, a mixture of higher aliphatic alcohols from hexanol to decanol is added in an amount of 3 moles per 1 mole phosphorous pentoxide. Aluminum sulfate is used as a crosslinker. Hexanol results in a high-temperature viscosity of the gel, while maintaining at a pumpable viscosity at ambient temperatures [870]. 

A particulate gel breaker for acid fracturing for gels crosslinked with titanium or zirconium compounds is composed of complexing materials such as fluoride, phosphate, sulfate anions, and multicarboxylated compounds. The particles are coated with a water-insoluble resin coating, which reduces the rate of release of the breaker materials of the particles so that the viscosity of the gel is reduced at a retarded rate [205]. 

In 1970, Monroe and Rooker(28) claimed the use of aluminum salts of acid orthophosphate esters as viscosity builders for use in fracturing fluids. The application of these materials began a new era of hydrocarbon gelling agents. Monroe(29) later claimed the use of Fe30it as a metal activator of phosphate esters and in 1971 described several other metals(30) that could be used with amine neutralization agents. Numerous metallic ionic derivatives can be used as effective "activators" or crosslinkers to prepare a gel. 

Purify the thiolated protein from excess DTT by dialysis or gel filtration using 50 mM sodium phosphate, 0.15 M NaCl, ImM EDTA, pH 7.2. The modified protein should be used immediately in a conjugation reaction to prevent sulfhydryl oxidation and formation of disulfide crosslinks. 

Dissolve a crosslinked protein or peptide that has been conjugated with the use of a disulfide-containing crosslinker at a concentration of l-10mg/ml in 0.01 M sodium phosphate, 0.15M NaCl, pH 7.4. Alternative buffer conditions and pH values may be used, however a pH between 7.0 and 8.1 usually works best. 

Dissolve the molecules to be conjugated in 0.1M sodium phosphate, pH 7.2 (for aqueous reactions) or in DMSO, DMAC, or methylene chloride (for organic reactions). If proteins are to be conjugated, a concentration of l-10mg/ml in buffer will work well in this protocol. For more dilute protein solutions, greater quantities of the bis-NHS-PEG compound may have to be added than recommended here to obtain similar levels of crosslinking. 

Dissolve the thiol-containing proteins to be crosslinked in 50 mM sodium phosphate, pH 6.5-7.5, containing 10 mM EDTA to prevent metal-catalyzed sulfhydryl oxidation. 

Remove excess crosslinker by gel filtration using a desalting resin. Perform the chromatography using 0.1M sodium phosphate, 0.15M NaCl, lOmM EDTA, pH 7.5. 

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