Glycine-hydrochloric acid buffer

The homogenization of liver, kidney, and muscle tissues with glycine-hydrochloric acid buffer was presented for the determination of OTC, TC, and CTC. The supernatant was purified on a cyclohexyl SPE cartridge previously activated with MeOH and water. The TCs were eluted with MeOH, which was evaporated at 65°C, and the residue was dissolved in mobile phase. Recoveries were achieved greater than 70% in muscle at the MRL concentrations and higher than 60% for kidney with RSD <11%. Gradient elution was employed to improve the separation of OTC and TC from interference found in kidney samples. The eluate from HPLC was mixed with aluminium chloride solution in a low-volume T-piece and delivered into the PTFE tubing 13.7 m X 0.3 mm immersed in an oil bath at 60°C followed by fluorescent detection (22). 

Glycine-Hydrochloric Acid Buffer (0.05 M) Dissolve 3.75 g of glycine in about 800 mL of water. Add 1 N hydrochloric acid until the solution is pH 3.0, determined with a pH meter. Quantitatively transfer the solution to a 1000-mL volumetric flask, dilute to volume with water, and mix. 

Sample Preparation Using Glycine-Hydrochloric Acid Buffer, prepare a solution of the sample enzyme preparation so that 2 mL of the final dilution will give a corrected absorbance of enzyme incubation filtrate at 275 nm (AA, as defined in the Procedure) between 0.200 and 0.500. Weigh the enzyme preparation, quantitatively transfer it to a glass mortar, and triturate with Glycine-Hydrochloric Acid Buffer. Quantitatively transfer the mixture to an appropriately sized volumetric flask, dilute to volume with Glycine-Hydrochloric Acid Buffer, and mix. 

Add 2 mL of Glycine-Hydrochloric Acid Buffer (instead of the Sample Preparation) to the substrate blank. After exactly 30 min, add 10 mL of TCA Solution to each enzyme incubation and to the substrate blank to stop the reaction. In the following order, prepare an enzyme blank containing 10 mL of Substrate Solution, 10 mL of TCA Solution, and 2 mL of the Sample Preparation. Heat all tubes in the water bath for 30 min, allowing the precipitated protein to coagulate completely. 

The chemical composition of the retrieval solution may affect the efficacy of the process and a wide variety of solutions have been advocated including citrate buffer, Tris buffer, glycine-hydrochloric acid, EDTA, urea, heavy metal solutions, and other proprietary reagents. The molarity of the solution may also significantly influence immunostaining (Taylor et al, 1996). 

The assay method of Dalziel is convenient. In a recording ultraviolet spectrophotometer set at 340 nm is placed a 3-mL quartz cuvette containing 2.4 mL of 0.10 M glycine-sodium hydroxide buffer solution, pH 9, 500 pL of a 54 mM solution of ethanol 1n the same buffer, and 100 pL of a 15 nM solution of NAD, also in the same pH 9 buffer. The volume is made up to 3.0 mL, and the assay initiated by the addition of 10 pL of a 1 mg per mL solution of HLADH in 0.10 M "Tris-hydrochloric acid buffer", pH 7.4. The change in optical density at 340 nm 1s monitored at 25°C and the activity calculated from the following equation ... 

Sample extraction/deproteinization is usually accomplished with mild acidic solvents to free the noncovalently bound tetracyclines from macromolecules. Mcllvaine buffer, pH 4.0 (286, 287), Mcllvaine/EDTA buffer, pH 4.0 (283, 287-293), succinate buffer, pH 4,0 (278-281,294-296), acidic acetonitrile (297-299), and acidic methanol (14, 199, 300) have all been used successfully. Moreover, trichloroacetic acid, pH 2.0 (301, 302), metaphosphoric acid (303), acetate buffer (126, 280), citrate buffer, pH 4.0 (304), citrate buffer/ethyl acetate, pH 4-5 (305), and hydrochloric acid/glycine buffer (306, 307) have all been employed with varying success to precipitate proteins from the sample homogenates. 

Glycine Buffer (0.2 M, pH 2.5) Dissolve 15.014 g of glycine (Merck, Catalog No. 4201) in about 800 mL of water. Adjust the pH to 2.5 with 1 M hydrochloric acid (consumption should be about 80 mL), and dilute to 1000 mL with water. 

Note Instead of the Glycine Buffer, some enzyme preparations may require the use of 0.01 M pH 8.0 Tris Buffer prepared as directed for Tris Buffer under Proteolytic Activity, Bacterial (PC), except to titrate with 1 N hydrochloric acid to pH 8.0. 

The Step 2 product was added to a 2.5mg/ml solution of Grob-t in Dulbeccu s phosphate buffered at pH 7.0 at a molar ratio of the Step 2 product/protein 2 1,4 1, or 10 1, respectively. The reaction was stirred for 3 hours at 40°C and quenched with 0.5 M glycine, and then the pH was lowered to 4.5 with 3 M of hydrochloric acid. The conjugate was isolated after purification by diafiltration. 

Many chemicals are available that can be used without further purification. Their selection is made on a trial and error basis, and even different lots from the same supplier may differ in purity. Volatile buffers and solvents such as pyridine, acetic acid, formic acid, and n-propanol, which are employed for chromatography, can be purified by distillation over ninhydrin. Constant-boiling hydrochloric acid is rountinely prepared over sodium dichromate (Schwabe and Catlin, 1974), and water of high purity is obtained from a system composed of a 0.2-fim particle filter, an activated charcoal cartridge, and two deionizer cartridges (Hydro Service and Supplies, Durham, North Carolina). The solvents are tested for purity in the following manner. A sample of the solvent is dried in vacuo. The residue is dissolved in pH 2.2 citrate buffer and injected onto the amino acid analyzer column. The distilled solvents are typically found to contain aspartic acid, serine, and glycine at 20-30 pmol/ml as the major contaminants. 

The buffer used in the sample is identical to that used in the stacking gel. The buffer used in the lower reservoir is identical to that used in the lower or separating gel. The buffer used in the upper reservoir is also an amine. It however, differs significantly from the rest of the buffers in one important way. Its pH. which is kept slightfy above that of the running gel. is adjusted not with hydrochloric acid, but with a weak acid whose pK is at the desired pH. Glycine is commonly used for this adjustment. 

Standard Solution. Weigh out a sufficient quantity of polymyxin and dilute it to contain 200 units per milliliter with glycine buffer prepared as follows glycine 3.6 g., sodium chloride 3.0 g., and distilled water 1 liter. Adjust the buffer to pH 2.0 with concentrated hydrochloric acid. 

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