Potassium phosphate buffer, solution preparation

An amount of enzyme preparation equivalent to 900 mg of wet cells was made up to 25 ml with the above potassium phosphate buffer solution. 150 mg (1.15 mmol) of 5-fluorouracil and 1.0 gram of thymidine (4.12 mmol) were dissolved in 15 ml of the above potassium phosphate buffer solution. The mixture was incubated at 37°C for 18 hours. After this time, enzyme action was stopped by the addition of four volumes of acetone and one volume of peroxide-free diethyl ether. The precipitated solids were removed by filtration, and the filtrate was evaporated under nitrogen at reduced pressure until substantially all volatile organic solvent had been removed. About 20 ml of aqueous solution, essentially free of organic solvent, remained. This solution was diluted to 100 ml with distilled water. 

Stock solution 5. 1 m stock solution of potassium phosphate buffer was prepared by dissolving K2HP04-3H20 (11.423 g) and KH2PO4 (6.805 g) in deionized water to a final volume of 100 mL. The pH was adjusted to 7.0. This 1 m stock solution was diluted to the desired concentration of 50 mM with deionized water. Buffers were stored at 0-4 °C. 

An amount of enzyme preparation equivalent to 900 mg of wet cells was made up to 25 ml with the above potassium phosphate buffer solution. 150 mg (1.15 mmol) of 5-fluorouracil and 1.0 gram of thymidine (4.12 mmol) were dissolved in 15 ml of the above potassium phosphate buffer solution. 

Prepare a solution of 5 m 3-cyanopyridine (320 mL) to be used as substrate for nicotinic acid preparation in 0.1 m potassium phosphate buffer pH 8.0. 

After preincubation of the brush border membrane vesicle preparation for 2 h, [2 14 C]urate uptake is initiated by adding 200 pi of incubation medium to 20 pi of the membrane suspension. The incubation medium has the following composition (mmol/1) 150 mannitol, 2 MgS04, 50 potassium phosphate buffer, pH 6.0 or 7.5, 0.02 [2-14 C]urate, and various concentrations of the inhibitor. At 10 s after the addition of the incubation medium, 200 pi portions of the suspension are pipetted onto the center of prewetted cellulose acetate filters kept under suction. The vesicles retaining on the filter are washed immediately with 5 ml of an ice-cold solution containing 150 mmol/1 mannitol and 50 mmol/1 potassium phosphate buffer, pH 6.0 or 7.5, which is used at the same pH as the incubation medium. Preincubations and incubations are performed at 23 1 °C. Each experiment is performed in triplicate. Corrections are made for the radioactivity bound to the filters in the absence of membrane vesicles. The term of the OH gradient-dependent urate uptake is defined as the difference between the uptakes in the incubation medium at pH 6.0 and that at pH 7.5. The OII gradient-dependent urate uptake at 10 s is assumed to present an initial velocity. 

Record the A340 of the solution at 30-sec intervals for 4 min. Determine whether the initial velocity of the reaction is linear over 4 min (AH340/Atime yields a straight line). If the kinetics are not linear over 4 min, dilute the enzyme twofold in 0.1 M potassium phosphate buffer, pH 7.5, and repeat the assay. Prepare a plot of A340 versus time and calculate the absolute value of the slope of the line. 

Dialyze the carrier protein against 10 mM potassium phosphate buffer, pH 7.2. At ust the carrier concentration to about 20 mg/mL. Weigh out the solid peptide and dissolve it in the same buffer at 5 mg/mL. Thiol-based scavengers, used to preserve free -SH groups in the peptide, are often present in peptide preparations and will need to be removed from the peptide solution by desalting on a Bio-Gel PIO column in 10 mM potassium phosphate buffer, pH 7.2. A brief nasal inspection is often sufficient to determine their presence. On the other hand, stock solutions of peptide (e.g., in PBS) may become oxidized if stored for extended periods, and so are best pretreated with 100 mMDTT (30 min at room temperature is adequate) to reduce the dissolved peptide before use. As the DTT will interfere with the conjugation, the reduced peptide should then be desalted on a PIO column as above. 

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. 

One to 2 ml of a freshly prepared red cell hemolysate is diluted to 50 ml in a final concentration of 1.5-2.0 mg/ml. Exactly 4.5 ml of hemoglobin solution is pipetted in test tubes labeled 0, 2, 4, 6, 8, 10, 15, 20, 30 minutes. Of a 2.5 M potassium phosphate buffer (pH 6.9), 0.5 ml is added to each tube. After mixing, the tubes are placed in a 60°C water bath. The tubes are removed from the water bath at the times indicated and placed immediately in an ice bath for 5 minutes. The... 

The protein 2-i mg/ml) is dissolved in 0.1 M potassium phosphate buffer, pH 7.5, containing 10 M EDTA. A 0.15 M solution of TNM is prepared in 95 % ethanol, and 20 pi is added per ml of protein solution (final TNM concentration 0.003 M). After 60 min at room temperature the reaction is terminated by the addition of 50 pi of 1 M mercapto-ethanol, followed immediately by exhaustive dialysis against 1 % NH4HCO3 at 4°C. The dialysis removes excess TNM and the colored nitroformate anion, which is a product both of the nitration reaction and of decomposition of the reagent. The dialyzed protein derivative is lyophilized. 

Substrate solution is prepared by dissolving 70 mg (2.3 mM) o-NP-G per 100 ml 100 mM potassium phosphate buffer, pH 7.0, containing ImM MgCh and 10 mM 2-ME. It is not advised to assay the enzyme over prolonged periods at temperatures exceeding 30°C because of possible spontaneous hydrolysis of the substrate (Section 10.1.3.5). At a fixed period after the addition of the substrate, catalysis is stopped with 0.25 volumes of 2 M Na2C03. The absorbance is measured at 405 nm, after calibration of the spectrophotometer... 

Prepare NADPH stock solution (20 mM) in water, test compound stock solution (20 mM) in appropriate solvent, / -(+ )-pulegone (Fig. 14.1) stock solution (20 mM) in water acetonitrile (1 1), dGSH stock solution (20 mM, Note 4), potassium phosphate buffer (0.1 M, pH 7.4), and chilled quenching solution (5 mM dithiothreitol in methanol). Human liver microsomes (20 mg/mL) are thawed on ice. 

Phosphate buffer (100 mM, pH 7.4) containing 1 mM EDTA was prepared by a dilution of 400 mM mono and dibasic potassium phosphate stock solutions (stored at 4°C) and stored at ambient temperature. Frozen stocks of HLM were used and the remaining was discarded after the first thawing. NADPH stock, at 10 mM in phosphate buffer was made fresh daily. Stock solutions of analytes (i.e., metabolites) were prepared in solvent and stored at —20°C or 4°C. Internal standards were dissolved in either DMSO or 50% acetonitrile in water. 

The reagent is prepared by the addition of a solution containing 70 mg of o-nitrophenyl-P-D-galactopyranoside (ONPG) per 100 mL of 0.1 AT potassium phosphate buffer, pH 7.0, containing 1 mM magnesium chloride and 0.01 AT2-mercaptoethanol. The reaction may be stopped by the addition of 0.25 vol of 2 AT sodium carbonate. 

Prepare in 50 mM potassium phosphate buffer, pH 7.4, an anaerobic solution of a ruthenate chelate, here potassium chloro[hydrogen(ethylenedinitrilo)nithen-ate, and dilute this in the same anaerobic buffer CO a final concentration of 50 j,m of the complex (Solution A). 

Standard Ctu-ve and Samples. Prepare a standard stock solution of 1 mg. per milliliter in 10% methanol-0.1 M potassium phosphate buffer, pH 7.0. This solution may be stored for one week at 5°C. Solutions are prepared for the standard curve by diluting the stock solution with phosphate buffer to final concentrations of 3.0, 2.0, 1.0, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, and 0.1 pg. per milliliter. Liquid samples are diluted with phosphate buffer on the basis of their estimated potencies to concentrations of about 0.6 Mg. per milliliter. Solid samples are prepared at 1 mg. per milliliter in 10% methanol-phosphate buffer and diluted as described above. 

A typical control solution contains 100 mM MOPS (3-(4-morpholino)-propanesulfonic acid), pH 7.6, 5 mM potassium phosphate buffer, pH 7.6, and 5 mM DFP or PMSF in a final volume of 8 ml. The assay solution is prepared by adding modified protein to an identical solution. 

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