2-Mercaptoethanol, solution preparation

We used an anti-DNA antibody as an exploratory model system. The antibody was monoelonal from mouse sourees and its subelass was IgM. Mouse IgG (MW 1.5 x 10 Da) and IgM (MW 9 X 10 Da) antibodies from normal plasma, and bovine serum albumin were used for the eontrol measurements. To prevent the nonspeeilie adsorption of proteins to the uneovered, bare Au site in the modified eleetrode surfaee, the DNA-modified eleetrode prepared by the standard proeedure was further treated with aqueous 2-mercaptoethanol solution and was used for the measurements. 

The specifications and standardization include raw materials, preparation method of the standard solution, concentration of proteins, and the main band on SDS-PAGE. The outline of the procedure for preparation of the calibrators is shovm in Eig. 4.2. Table 4.5 shows the raw materials and the preparation method of the initial extract. To prepare the calibrators, the raw materials are extracted by the standard solution containing SDS and mercaptoethanol. The initial extract is prepared by centrifugation and filtration of the extract. The diluted extract is then prepared by 10-fold dilution of the initial extract with phosphate-buffered saline (PBS pH 7.4). The protein concentration of the diluted extract is assayed using the 2-D Quant kit (Amersham Bio Sciences). The standard solution is then... 

To prepare 10-formyl TF1F (formyl donor for the formylation of fMet-tRNAfMet), 10 mg of 5-formyl-tetrahydrofolate (5-formyl THF) are dissolved in 1 ml of 0.1 MHC1 that has been flushed extensively with N2 and kept overnight at 4° in a closed vial. The resulting yellow suspension of 5,10-methenyl THF is agitated and an aliquot withdrawn for spectropho-tometric determination of the concentration (6 mM solution = 85 A355). The expected 5,10-methenyl THF concentration is approximately 20 mM. The cyclic methenyl THF can be stored for several weeks at —20°. The 10-formyl THF is stable for only a few weeks and is freshly prepared hydrolyzing the cyclic methenyl THF. For this purpose, the stock solution is diluted in 100 mM Tris-HCl (pH 8.0) extensively flushed with N2 and supplemented with 100 mM 2-mercaptoethanol. After 15 min incubation at 20°, the solution is divided in aliquots, which are stored at —20°. 

Prepare a solution consisting of 2.5 ml glycerol, 100 pi of 10 percent Triton X-100 (Thermo Fisher Surfact-Amps X-100), and 10 pi 2-mercaptoethanol. 

OPA 97% (Aldrich) prepared by dissolving 20 mg of the salt in 0.5 ml methanol. Add to this solution 3 ml of the potassium borate buffer and 2 ml H20, followed by 30 pi mercaptoethanol. This reagent is stable for 1 week when stored in the dark. 

Method (manual). An aliquot portion (0.1 ml) of effluent containing the amino acid is mixed with 3 ml of a solution containing 1.5 ml of o-phthalaldehyde (10 mg/ml in ethanol), 90 ml of sodium tetraborate buffer (0.05 M, pH 9.5) and 1.5 ml of a solution of 2-mercaptoethanol (5 mg/ml) in ethanol. The latter reagent should be freshly prepared each day. The reaction mixture is permitted to stand for 5 min and the intensity of fluorescence is measured in a fluorimeter (340-nm excitation, 455-nm emission). The buffered reagent may be also useful as a spray reagent for amino acids separated by TLC, although such an investigation has not been reported. 

General procedure for the preparation of oxathiolanes.140 To a stirred refluxing solution of cycloheptanone (56.1 g, 0.5 mol) and 2-mercaptoethanol (29.1 g, 0.5 mol) in anhydrous ether (400 ml) was added dropwise over a 1-hour period boron trifluoride-etherate (71 g, 0.5 mol). After an additional hour of being heated under reflux, the solution was allowed to cool, washed with 0.1 m sodium hydrogen carbonate solution (2 x 100 ml) and once with saturated sodium chloride solution (100 ml), and dried over magnesium sulphate. After removal of the solvent under vacuum on a rotary evaporator, the residue was distilled under vacuum to yield a small forerun which was followed by 2-oxa-5-thiaspiro[4.6]undecane (79.2g, 92%), b.p. 77-78°C/1.2mmHg, n 5 1.5165. 

Carbamate pesticides are best analyzed by HPLC using postcolumn deriva-tization technique. Some common carbamate pesticides are listed in Table 2.19.1. Compounds are separated on a C-18 analytical column and then hydrolyzed with 0.05 N sodium hydroxide. Hydrolysis converts the carbamates to their methyl amines which are then reacted with o-phthalaldehyde and 2-mercaptoethanol to form highly fluorescent derivatives. The derivatives are detected by a fluorescence detector. o-Phthaladehyde reaction solution is prepared by mixing a 10-mL aliquot of 1% o-phalaldehyde solution in methanol to 10 mL of acetonitrile containing 100 pL of 2-mercaptoethanol and then diluting to 1 L with 0.05 N sodium borate solution. 

The reaction mixture included phosphate buffer, alcohol dehydrogenase, NAD+, substrates and internal standards, and mercaptoethanol in a final volume of 190 gtL. The enzyme preparation is preincubated with all components of the assay system except substrates, which are added to initiate the reaction. The reaction is terminated by adding 100 giL of the incubation mixture of 300 fiL of reaction-terminating solution containing acetate-perchlorate buffer and ascorbic acid. Samples could be stored up to 2 weeks at -80°C before thawing and filtering through 0.45 gm nitrocellulose filters. Flve-microliter aliquots were injected into the HPLC... 

Sample preparation solution To 10 mL of stacking gel buffer, add 2 g of SDS, 5 mL of 2-mercaptoethanol, 10 mL of glycerol, and 5 mg of bromophenol blue. Make up to a final vol of 100 mL with distilled water. Store at room temperature. 

Cysteine residues in transferrin were reduced and alkylated in a similar manner as that done for solution samples p). Modiflcation for samples prepared with ProSorb cartridge can be performed in the same ProSorb cartridge before membrane removal, while modifications were performed in an Eppendorf tube for the electroblotted samples. The membranes were incubated 15 minutes at room temperature in a 0.25 M Tris/HQ and 6 M Guanidine hydrochloride buffer containing 1 ml of mercaptoethanol and followed by the addition of 1 ml of 4-vinyl pyridine for another 15 minutes. The membnmes were washed thoroughly with 0.1% TFA afterwards. 

Before crystallization trials, the protein was subjected to gel filtration on Superdex-75 (Pharmacia) in 50 mM sodium/potassium phosphate buffer, pH 7.4, containing 1 mM EDTA, 50 mM 2-mercaptoethanol, 150 mM sodium chloride, 5% glycerol and 5% 2-propanol, as described previously (12). The statine-based inhibitor, LP-149 (Ac-Nal-Val-Sta-Glu-Nal-NH2 e Nal is naphtylalanine and Sta is statine) (Fig. 1), was prepared at Lilly Research Laboratories (K. Hui, unpublished results). Crystallization was carried out at 4 °C using the hanging-drop vapor diffusion method as follows 2.5 //I of the FIV PR(D30N) at 7 mg/ml complexed with LP-149 (1 4 molar ratio) in 50 mM imidazole-HCl pH 7.0 containing ImM EDTA and 1 mM dithiothreitol were mixed with an equal volume of 2 M ammonium sulfate, 0.1 M sodium acetate, pH 4.6 (Hampton Crystal Screen, solution 47). Crystals appeared within a few days and reached the size of 0.2 x 0.2 X 0.4 mm in one week. 

By y-ray irradiation at room temperature, ZnS nanowires were grown, in which the crystal growth was in situ templated by an inverted hexagonal liquid crystal formed from oligo(ethylene oxide)oleyl ether amphiphiles, n-hexane, n-hexanol/ isopropanol (2/1) and water [151]. Nanocrystalline jS-ZnS (sphalerite) was prepared in aqueous isopropanol solution [152]. The TEM images (Figure 7.36) indicate that the product from mercaptoethanol is most well dispersed, and that from thiourea... 

---