Cacodylate buffer

Cacodylate buffers Cacodylate (pH 5.5—7.0) has been used in the past for spectroscopic studies with nucleic acids. Being a compound of arsenic, it is toxic It reacts with SH groups, which disqualifies it for studies of proteins and enzymes. 

The rates of hydrolysis and binding to DNA of anti-DE-I, syn-DE-I, anti-DE-II, syn-DE-II, and anti-1,2-dihydroxy-3,4-epoxy-1,2,3,4-tetrahydrochrysene (anti-chrysene-DE) were studied in order to relate the chemical reactivity of these dihydrodiol epoxides to their biological activities. The half-lives of the dihydrodiol epoxides in cacodylate buffer at pH 7.0 and 37°C are summarized in Table III and their relative extents of binding to DNA in Table IV. It is clear that the rates of hydrolysis of the dihydrodiol epoxides do not correlate with their DNA binding properties. 

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). 

The experimental observations in cacodylate buffer solutions are consistent with a mechanism involving a kinetically common intermediate according to the following reaction scheme ... 

Tannic acid can be added to the prefixative (1—4% w/v) in cacodylate buffer, or tannic acid in 0.1 M buffer 1-2 h can be used after osmication between fixation and dehydration. 

Tannic acid Tannic acid can be added at 1-4% w/v in either cacodylate-buffered glutaraldehyde or glutaraldehyde-formaldehyde or between prefixation and postfixation (immerse specimens 1-2 h in tannic acid in 0.1M buffer) Tonoplast... 

Two-thirds of the blocks of 1 mm diameter were immersed for 12 h in a ZIO mixture prepared according to the method of Maillet (1), and was first utilized in electron microscopy by Stach (15a). The remaining blocks were prefixed at 4°C for 2 h in 0.1 M cacodylate buffer containing 5% glutaraldehyde for 15 min, and immersed in the ZIO mixture at 25°C for 12 h. The ZIO mixture was prepared as follows twelve to 15 g of zinc (powder) and 5 g of iodine (crystal) were dissolved in 200 mL of distilled water. Eight milliliters of the filtered solution was added to 2 mL of 2% Os04 solution prior to use. 

The tissues were fixed in 0.05 M cacodylate buffer containing 2.5% glutaraldehyde and 1.5% formaldehyde (pH 7.0) for 16 h. The ZIO mixture was prepared as follows 3 g zinc (powder) and 1 g resublimed iodine crystals were dissolved in 20 mL distilled water. After stirring for 5 min, the zinc was filtered off. The filtered solution was mixed with an equal volume of 2% 0s04 solution and the solution used immediately. Treatment with the ZIO mixture was carried out for 4 h at room temperature. 

Samples are ready for treatment, if any. In this case, samples were washed in a buffer solution of 0.1 MNa-cacodylate, pH 7.4,4% sucrose, 1 mM PMSF and various concentrations of divalent cations. Incubation is for 10 min at 4°C with three rapid changes. 

Fix with 4% glutaraldehyde buffered with 0.1 MNa-cacodylate, pH 7.2 for 24 h in a refrigerator (the solution in this case also contained the appropriate concentration of divalent cations). 

Washed cells with CKM buffer (0.05 MNa-cacodylate, 0.025 MKC1,... 

Tissue processing tissue specimen (0.5 1.0 mm3) are fixed in 4% buffered formalin for 30 60 min, post-fixed in 1% osmium tetroxide in cacodylate or phosphate buffer, pH 7.2 7.4, stained en bloc for 30 min with 2% aqueous uranyl acetate, then dehydrated in ethanol and embedded in epoxy or acrylic resin. 

A similar transition induced by changing the buffer from 10 mM cacodylate to 10 mM Tris is observed in some, but not all, samples of pBR322 DNA... 

Fig. 6. Effect of the concentration of camphor and the ionic strength on the fraction of bacterial cytochrome / -450 in the high-spin state (Xhs) in aqueous solution at pH 7 in 25 mAf sodium cacodylate buffer at 18°C. , No added KCl 9. 50 mAf KCI.

Fig. 11. A plot of proton activity pan as a function of electromotive force (mV) for the ethylene glycol-glass electrode at +21°C and — 19°C. The response of the electrode (R ) is given by the slope of the line. The concentration of ethylene glycol is 50% by volume. The points are the experimental results in different buffer systems (a, chloroacetate b, acetate c, cacodylate d, Tris) the straight lines represent ideal behavior.

Scheme 10.3 Ceramide glycanase mediated release by transglycosylation. a) Pd/C, MeOH b) (8), EEDQ, EtOH-CgHe c) MeONa d) CH2=CHCONH2, TMEDA, APS, DMSO-H2O, 50"C, CMP-NeuAc, a-2,3-sialyltransferase, BSA, MnCb, CIAP, 50 nM sodium cacodylate buffer, pH 7.49, ceramide, Triton CF-54, sodium citrate buffer, pH 6.0, 37°C, 61%.

Microscopic analysis. From each experimental group of specimens with incipient lesions, two specimens were selected randomly for microscopic analysis. Two adjacent slices (500 pm thickness) were prepared from each specimen with a diamond wire sectioning machine (model 3242, Well, Le Lode, Switzerland). The slices were fixed in 1.0 ml 4% paraformaldehyde, 1% glutardialdehyde in 0.1 M sodium cacodylate buffer, pH 7.4, for one week. One slice of each pair was subsequently demineralized in 2.0 ml 0.1 M acetic acid, 2.5% glutardialdehyde, pH 4.0. 

Specimens were post-fixed in 1% OSO4 in sodium cacodylate buffer, dehydrated in ascending alcohol solutions, and embedded in Epon LX 112. Semi-thin sections (1.0 pm thickness) were cut with a diamond knife and stained with Richardson s dye (contains methylene blue) or PAS-reagent. Micrographs were made with an Olympus New Vanox microscope. 

Equilibration buffer contains potassium cacodylate (dimethylarsinic acid). Avoid contact with skin and eyes. Harmful if swallowed. Wear appropriate protective clothing. 

O.IM Cacodylate buffer 21.4 g cacodylic acid 3 H2O, sodium salt bring volume to 1 L with deionized glass-distilled water adjust pH to 7.4 with HCl. 

Samples may be fixed in 2% OSO4 in O.IM cacodlylate buffer for 1 h at room temperature and then rinsed in O.IM cacodylate buffer prior to embedding. 

Immediately after irradiation quench the samples by rinsing them with PBS or O.IM cacodylate buffer at 4°C. 

Clean the vial and score it with a diamond scribe. Place it in a clean glass bottle, and add the water. With a glass rod, break the vial. The solution is stable if kept refrigerated and protected from light. The stock solution should be stored away from other chemicals, since osmium vapors may escape from the bottle. Alternatively, aqueous solutions of osmium tetroxide in sealed glass ampules are available commercially. Dilute them to 1-2% with O.IM cacodylate buffer for use. 

For better morphological preservation, refix the cells in 2% glutaraldehyde-2% formaldehyde in 0.1 M cacodylate buffer, pH 7.4 for 1 h at room temperature. 

Rinse in cacodylate buffer, postfix in 1-2% osmium tetroxide, and embed as usual (see Chapter 40 Note 9). 

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