Other Buffers

Because of the well-known abnormalities of the liquid junction potentials when making pH measurements at the extremes of the pH scale, the tetroxalate and calcium hydroxide buffers are regarded as secondary. Both are sensitive to dilution, but only pH 12.45 buffer is highly sensitive to temperature changes. 

Many other buffers have been developed for various purposes. Generally, these alternate compositions are less stable or accurate than the primary buffers described the the National Bureau of Standards. They are often employed when special conditions must be met or when the buffer is more compatible with the sample. Some of these buffers are described in Section 4.4. One scheme of buffer compositions which cover the pH range 1-10 has been developed by Clark and Lubs. These are described in Table A.3 and, generally, with others listed in the Handbook of Biochemistry [5]. 

Standardization of Acidity Measurements, Anal. Chem. 40, 28A (1968). 

Handbook of Biochemistry, 2nd ed.. Molecular Biology, p. J195. Chem. Rubber Co., Cleveland, Ohio, 1970. 

Perrin, D. D., and Dempsey, B., Buffers for pH and Metal Ion Control. Halsted Press, New York, 1974. 

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Table 8.19 pH Values of Biological and Other Buffers for Control Purposes 8.110... 

Developing agents must also be soluble in the aqueous alkaline processing solutions. Typically such solutions are maintained at about pH 10 by the presence of a carbonate buffer. Other buffers used include borate and, less frequendy, phosphate. Developer solubiUty can be enhanced by the presence of hydroxyl or sulfonamide groups, usually in the A/-alkyl substituent. The solubilization also serves to reduce developer allergenicity by reducing partitioning into the lipophilic phase of the skin (46). 

One mol of 2,6-xylidine is dissolved in 800 ml glacial acetic acid. The mixture is cooled to 10°C, after which 1.1 mol chloracetyl chloride is added at one time. The mixture is stirred vigorously during a few moments after which 1,000 ml half-saturated sodium acetate solution, or other buffering or alkalizing substance, is added at one time. The reaction mixture is shaken during half an hour. The precipitate formed which consists of cj-chloro-2,6-di-methyl-acetanilide is filtered off, washed with water and dried. The product is sufficiently pure for further treatment. The yield amounts to 70 to 80% of the theoretical amount. 

EFFECTS OF OTHER BUFFER FORMULATIONS ON RECOVERY EFFICIENCY... 

The experimentally observed pseudo-first order rate constant k is increased in the presence of DNA (18,19). This enhanced reactivity is a result of the formation of physical BaPDE-DNA complexes the dependence of k on DNA concentration coincides with the binding isotherm for the formation of site I physical intercalative complexes (20). Typically, over 90% of the BaPDE molecules are converted to tetraols, while only a minor fraction bind covalently to the DNA bases (18,21-23). The dependence of k on temperature (21,24), pH (21,23-25), salt concentration (16,20,21,25), and concentration of different buffers (23) has been investigated. In 5 mM sodium cacodylate buffer solutions the formation of tetraols and covalent adducts appear to be parallel pseudo-first order reactions characterized by the same rate constant k, but different ratios of products (21,24). Similar results are obtained with other buffers (23). The formation of carbonium ions by specific and general acid catalysis has been assumed to be the rate-determining step for both tetraol and covalent adduct formation (21,24). 

Remove unreacted N-acetyl homocysteine thiolactone and reaction by-products by gel filtration or dialysis against lOmM sodium phosphate, 0.15M NaCl, lOmM EDTA, pH 7.2. Other buffers suitable for individual protein stability may be used as desired. For the silver nitrate-containing reaction, removal of the silver-thiourea complex may be done by adsorption onto Dowex 50, and the protein subsequently eluted from the resin by 1M thiourea. Removal of the thiourea then may be done by gel filtration or dialysis. 

Dissolve the protein to be reduced at a concentration of l-10mg/ml in 20 mM sodium phosphate, 0.15M NaCl, pH 7.4. Other buffers and pH values also may be used. A strong denaturant may be added (6M guanidine or 8M urea) to this solution to promote protein unfolding and make buried disulfides more accessible. 

Dissolve the protein to be modified in 50mM sodium borate, pH 8.5, at a concentration of lOmg/ml. Other buffers may be used for an NHS ester reaction, including 0.1M sodium phosphate, pH 7.5 (Chapter 2, Section 1.4). 

Dissolve the antibody to be biotinylated in 50 mM sodium bicarbonate, pH 8.5, at a concentration of 10 mg/ml. Other buffers and pH conditions between pH 7 and 9 can be used as long as no amine-containing buffers like Tris are present. Avoid also the presence of disulfide reducing agents that can cleave the disulfide group of the biotinylation reagent. 

The following protocol should be compared to the method described for SATA thiolation in Chapter 1, Section 4.1. Although the procedures are slightly dissimilar, the differences indicate the flexibility inherent in the chemistry. For convenience, the buffer composition indicated here was chosen to be consistent throughout this section on enzyme-antibody conjugation using SMCC. Other buffers and alternate protocols can be found in the literature. 

Dissolve the protein to be modified with TsT-mPEG in ice-cold 0.1 sodium borate, pH 9.4, at a concentration of 2-10 mg/ml. Other buffers at lower pH values (down to pH 7.2) can be used and still obtain modification, but the yield will be less. Avoid amine-containing buffers such as Tris or the presence of sulfhydryl-containing compounds, such as disulfide reductants. 

Washes and dilutions wash sections in 10 mM sodium phosphate buffer, pH 7.5, 150 mM NaCl (PBS) for 2 x 3 min. PBS is used for all washes and dilutions. Other buffers such as Tris buffered saline (TBS) may also be used. 

Other buffers that have been used for continuous, native electrophoresis are Tris-glycine (pH range 8.3 to 9.5),19 Tris-borate (pH range 8.3 to 9.3),26 and Tris-acetate (pH range 7.2 to 8.5).27 Borate ions26 can form complexes with some sugars and can therefore influence resolution of some glycoproteins. 

The sodium acetate-acetic acid combination is one of the most widely used buffers, and is usually referred to simply as acetate buffer. Other buffer combinations commonly employed in chemistry and biochemistry include carbonate-bicarbonate (sodium carbonate-sodium hydrogen carbonate), citrate (citric acid-trisodium citrate), phosphate (sodium dihydrogen phosphate-disodium hydrogen phosphate), and tris [tris(hydroxymethyl)amino-methane-HCl]. 

There are several possibilities for the determination of the critical micellar concentration. If the micelles are formed from charged surfactants, a plot of the electrophoretic current at constant high voltage against the surfactant concentration shows an inflection point at the ccmc. It should be noted that the critical micellar concentration changes with temperature, the kind and concentration of counterions, and other buffer ingredients. 

Equations (49) to (52) are derived for the simple case in which only one surfactant forms one type of micelles. Depending on surf actant concentration and other buffer constituents, a more or less complicated mixture of micelles differing in form and/or composition coexist in the buffer. All of these micelles may exhibit different partition behavior in relation to a certain... 

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