Dissolved 2-mercaptoethanol

A sample of aequorin (purity > 80%) is first luminesced by adding a sufficient amount of Ca2+. To the spent luminescence solution, ammonium sulfate is dissolved to a concentration of 1M, and then the solution is added onto a column of Butyl Sepharose 4. The apoaequorin adsorbed on the column is eluted stepwise with buffer solutions containing decreasing concentrations of (NH4)2S04 starting from 1M. Apoaequorin is eluted at a (NH4)2SC>4 concentration lower than 0.1 M. The apoaequorin eluted is regenerated with coelenter-azine in the presence of 5 mM EDTA and 2 mM 2-mercaptoethanol... 

Purified LBP is obtained from the crude LBP separated in the gel filtration of the 35 kDa luciferase on Sephadex G-100 (see Fig. 8.2). The fractions of crude LBP are combined and the protein is precipitated with ammonium sulfate (75% saturation). The precipitate is dissolved in a small volume of lOmM Tris-HCl/5 mM 2-mercaptoethanol, pH 8, and a small amount of luciferin is added as a tracer. Then, the crude LBP is purified on a column of Sephadex G-200 (Hastings and Dunlap, 1986). The fractions of LBP are identified by luminescence produced by the addition of luciferase at pH 6.3 the luminescence due to the tracer luciferin is proportional to the amount of LBP in each fraction. 

Merino-Merino et al. [32] used the OPA reagent (o-phthaldehyde condensed with 2-mercaptoethanol) to separate penicillamine enantiomers after their derivatization. Racemic and (/q-penicillamine were dissolved in aqueous 0.5 M NaOH, and treated with the derivatizing solution (methanolic o-phthaldehyde and 2-mercaptoethanol in 0.4 M potassium borate buffer solution of pH 10). The reaction mixture was set aside for 2 min at room temperture, whereupon a portion of solution was analyzed by HPLC. The method used a Cyclobond column (25 cm x 4.6 mm) maintained at 5 °C, a mobile phase of ethanol/1% triethylammonium acetate (1 1 pH 4.5) eluted at... 

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 The resulting yellow suspension of 5,10-methenyl THF is agitated and an aliquot withdrawn for spectropho-tometric determination of the concentration (6 mM solution = 85 A355).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°. 

The cells are lysed in a buffer containing strong chaotropic reagents such as guanidine thiocyanate and 2-mercaptoethanol, which completely denatures any ribonuclease present. The supernatant is then placed on a cushion of CsCl (5.7 mol l-1) and centrifuged at 100000 g for 18 h. The RNA passes through the CsCl and is pelleted, while the DNA and protein remain in the aqueous solution. The RNA pellet is dissolved in buffer and concentrated by precipitation in cold ethanol. 

A-Vinyl-2-pyrrolidone (1.8 mol), 2,2 -azobis(2-methyl-N-(2-hydroxyethyl)-propio-namide) (0.018 mol), and 2-mercaptoethanol (0.072 mol) were dissolved in 3000 ml of 2-propanol degassed for 1 hour. The free radical polymerization was carried out at reflux while stirring for 44 hours. The mixture was concentrated and then dissolved in 400 ml of 4-methyl-2-pentanone and slowly precipitated into 5000 ml of t-butyl methyl ether. The suspension was filtered and the filter cake washed twice with 200 ml of -butyl methyl ether. The solid was purified by dissolving in 400 ml of... 

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. 

Upon oxidation these dithiols cyclize to form stable disulfides, driving the reaction to completion. These same compounds are also widely used to protect SH groups in enzymes against accidental oxidation by oxygen and to dissolve highly crosslinked insoluble proteins. Mercaptoethanol, HS-CH2-CH2-OH, may be used for the same purposes but requires higher concentrations and has a disagreeable odor. 

Substrate solution for (3-galactosidase- 4 mg/mL dinitrophenyl-P-D-galactopy-ranoside (ONPG) dissolved in TBS containing 0.7% 2-mercaptoethanol and 1 mMMgCl2... 

Dissolve the macromolecule containing sulfhydryl groups to be blocked in a buffer having a pH of 6.5—7.5. Sodium phosphate (0.01—0.1 M) at pH 7.2 works well. Avoid amine-containing buffers, since an excess of amines may cause some reactivity with the maleimide groups. Also, avoid the presence of sulfhydryl-containing disulfide reductants such as DTT or 2-mercaptoethanol, which will rapidly react with NEM. 

Despite the assertion over a number of years that vanadate is rapidly reduced in the presence of ligands such as p-mercaptoethanol and dithiothreitol, it is now known that this assertion is subject to qualification. Solution studies have shown that the vanadium(V) complexes of p-mercaptoethanol [36] and dithiothreitol [37] can be quite stable in solution. At pH 7.1, reduction of vanadium in the dithiothreitol complex occurs in a timescale of about 90 min, and even at pH 6.2, significant production of V(IV) is not observed for about 20 min. When the crystalline vana-dium(V) complex of p-mercaptoethanol was dissolved in water, it was shown to... 

The effect of dissolved oxygen, free S02, pH, and time/temperature influences on the content of dimethyl sulfide (DMS), 2-mercaptoethanol, dimethyl sulfone, methional, and 3-(methylthio)-l-propanol in ports was studied by Silva Ferreira et ah (2003a). They found that 3-(methylthio)-l-propanol decreased significantly in the presence of O2, and no methional formed. 2-Mercaptoethanol also decreased in the presence of 02, whereas... 

In reactions run with mercaptans, either 1.0 g of 1-hexanethiol or 0.1 g of 2-mercaptoethanol was dissolved in 15 g of acrylonitrile. The reaction mixture was stirred magnetically and irradiated with cobalt-60 to a total dose of 0.1 Mrad. 

Figure 9.1 shows the fluorescence spectrum of tryptophan residues of cytochrome b2 core dissolved in 6 M guanidine, pH 7, in the presence of 30 mM 2-mercaptoethanol. The emission peak is located at 357 nm, indicating that the protein is completely denatured (spectrum a). The fluorescence emission spectra of successive aliquots (0.55 /uM) of free tryptophan added to the protein solution are also shown (spectra b-g). 

X SDS/EDTA buffer—Dissolve 3.51 g of Tris HC1 and 0.335 g of Tris free base in 50 ml of 0.5 M EDTA, pH 8.0. Adjust the pH to 7.0 with dilute NaOH or HC1 if necessary. Add 30 ml of glycerol, 10 ml of /3-mercaptoethanol, and 10 ml of distilled water. Dissolve (avoid foaming) 8 g of SDS into this solution, as well as 4 mg of bromophenol blue. Store the solution tightly capped at room temperature. 

A 0.535-g (1 mmole) portion of [Pt(terpy)Cl]Cl-2H20 is dissolved in about 20 mL of deionized water in a 50-mL beaker, which is then covered. The red-orange solution is stirred and continually flushed with N2 at room temperature. Pure 2-mercaptoethanol (80 pL, 1 mmole) is added slowly, and the dark-red... 

---