Dithiothreitol, solution preparation

The SI70 supernatant (220 ml) was made to 40 % saturation with solid ammonium sulfate, stirred for 20 min, and then the precipitate was collected by centrifugation at 15,000 g for 15 min. The precipitate was suspended in small volume of buffer B-50 at pH 7.6 containing 20 mM HEPES/KOH, 0.1 mM EDTA, 1 mM dithiothreitol, 10 % (v/v) glycerol, and 50 mM potassium acetate. The 60 % saturated ammonium sulfate solution was prepared similarly. Protein concentrations for 0 - 40 % and 40 - 60 % ammonium sulfate fractions were 4.2 mg/ml and 4.7 mg/ml, respectively. 

Dithiothreitol (DTT) (Cat. No. 20710, Serva, Heidelberg, Germany) molecular weight 154.3 Prepare 10 mM solution fresh before use dissolve 1.54 mg DTT in 10 ml demineralised water. 

Triton X-100, EDTA, dithiothreitol and electrolyte protect enzyme in dilute solution and against denaturation by heat or extreme pH-values [12, 48] <2>, at low dithioerythritol concentrations enzyme tends to aggregate [5] <2>, bovine serum albumin, 1 mg/ml, stabilizes dilute enzyme solutions [5] <2>, diadenosine pentaphosphate, i.e. AP5A, stabilizes during preparative electrophoresis [7]... 

Microcystin-LR (MC-LR) (Sigma, France) standard solutions first prepared in 50 50 methanokwater and subsequently diluted in 30 mM tris-HCl, 2mM dithiothreitol (DTT), 2mM ethylene diamine tetraacetic acid (EDTA), 0.2mgmL 1 bovine serum albumin (BSA), 20 mM MgCl2 buffer (pH 8.4) (prepared in Milli-Q water). 

DTT solution Dissolve 15.42 mg 1,4-dithiothreitol (>99% p.a Carl Roth GmbH Co. KG, Karlsruhe, Germany store at 4°C) in 100 p,L water. Prepare fresh for each reaction. Do not store. 

Mix 9 microlitres (0.1 M Tris-Cl pH 7.4, 0.1 M MgCl2 0.5 M NaCl)+ 1 microlitre (0.1 M DTT) (dithiothreitol). Keep stock solutions frozen, prepare mixture immediately before use. 

A second type of rapid preparation that can be used to prepare DNA successfully from small amounts of starting material22 is to lyse an extremely small amount of cell suspension (usually less than 10 /d) by adding 5 pi of 200 mM potassium hydroxide, 50 mM dithiothreitol and incubating at 65° for 10 min. Five microliters of neutralization solution (900 mM Tris-HCl, pH 8.3, 300 mM potassium chloride, 200 mM hydrogen chloride) is then added. This solution should then be ready for PCR amplification. 

Four recent examples of universal linkers/supports, in which the first nucleoside is anchored onto the preformed linker-support construct, are shown in Fig. 2.14. The disulfide linker 2.33 has been used to prepare terminal 3 -phosphate ONs (94, 95) through cleavage with a solution of ammonia in dithiothreitol. The photolabile linker 2.34 (96) is used to prepare 3 -alkyl carboxylic acids. The allyl-based linker 2.35 (97) is used to prepare free 3 -OH ONs by cleavage with Pd(0) and treatment with an aqueous buffer at pH 10. The linker 2.36 (98) differs from those discussed so far in... 

Extraction buffer 1% SDS, 1% aprotinin, 0.5 mMphenylmethylsulfonyl-fluoride (PMSF),and50mAfdithiothreitol inTris-bufferedsaline (TBS 144 mMNaCl, 25 mAfTns, pH 7.6). Aprotinin is a protease inhibitor obtainable in solution from Sigma the stock solution is diluted 1 100. PMSF, a protease inhibitor, is toxic and can be omitted in many instances. It is added from a 100 mMsolution in isoamyl alcohol. Dithiothreitol is added to the extraction buffer immediately before use. For some purposes, the assay buffer is prepared at 2X normal strength by doubling the concentrations of SDS, aprotinin, and PMSF in TBS. 

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. 

In addition to the buffers needed for cell permeabilization prepare Solution LC supplemented with the required amount of TeTx. If the whole toxin (heavy plus light chains) is used, the catalytic subunit must be separated from the heavy chain by dithiothreitol (DTT) treatment. We usually incubate a 200-fold concentrated toxin in LC supplemented with DTT (final concentration 10 mM) for 30 min at 37°C. If the light chain is used this activation step is unnecessary. 

Enzyme stock solution can be purchased from Bachem Bioscience (Torrance, CA) or prepared as described (see ref. 10). The author s stock solutions typically contained 100 mil sodium MES, pH 6.5,10% glycerol, 5% ethylene glycol, and 50 voM dithiothreitol (DTT) and were stable for more than 1 yr at -70°C. Stock... 

Add 1 mL of 0.2 M dithiothreitol prepared in 1M acetic acid. Vortex solution at room temperature for 3h. 

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