Phosphate buffer washes

The inside capillary wall controls the electroosmotic velocity and provides undesired adsorption sites for multiply charged molecules, such as proteins. A fused-silica capillary should be prepared for its first use by washing for 15 min each (> 20 column volumes) with 1 M NaOH and 0.1 M NaOH, followed by run buffer ( —20 mM buffer). For subsequent use at high pH, wash for 10 s with 0.1 M NaOH, followed by deionized water and then by at least 5 min with run buffer.28 If the capillary is being run with pH 2.5 phosphate buffer, wash between runs with 1 M phosphoric acid, deionized water, and run buffer.29 When changing buffers, allow at least 5 min of flow for equilibration. For the pH range 4-6, at which equilibration of the wall with buffer is very slow, the capillary needs frequent regeneration with... 

Although the HRP/TMB system is usually a good, reliable, and sensitive combination, HRP has a number of alternative substrates, which can be used such as o-phenylene diamine. There are also number of options for the enzyme used other than HRP, such as alkaline phosphatase, which can be used in combination with the substrate p-nitrophenyl phosphate. It is important to note that if alkaline phosphatase is used, the wash buffer must not contain phosphate. Usually in this case a Tris-buffered rather than phosphate-buffered wash buffer is used. The choice of enzyme-substrate system depends on a number of factors, including price, sensitivity and whether a spectrophotometer filter is available for the substrate specific wavelength to be measured. 

Rc measures the extent to which a plateau has been attained. To make R< = 1 under conditions of strictly linear, i.e., noncurving kinetics, the denominator is multiplied by 3, i.e., by the number of phosphate buffer washes. As a plateau is approached, increases in value and in the limit approaches infinity when perfect plateau conditions have been attained. 

Sample preparation Condition an AASP C8 SPE cartridge (Varian) by washing with two 1 mL portions of MeCN and two 1 mL volumes of 67 mM pH 7.0 buffer. 150 p,L Plasma + 1.5 mL 67 mM pH 7.0 phosphate buffer -t- 20 p,L water, add a 1 mL aliquot to the SPE cartridge, wash with 200 p.L 67 mM pH 7.0 phosphate buffer, wash three times with 200 mg/L sodium azide in 67 mM pH 7.6 buffer. Place SPE cartridge on-line so that it is eluted onto the anal3dical column. 

Sample preparation Condition a 2.8 mL 500 mg Bond Elut C18 SPE cetrtridge with 4 mL MeOH and 1 mL 50 mM pH 6.8 phosphate buffer. 200 piL Plasma or 50 p,L middle ear fluid -I- 35 pL 60 pg/mL cefadroxil in MeOH water 5 95 -b 1 mL 50 mM pH 6.8 phosphate buffer, vortex, add to the SPE cartridge, wash with 1 mL 50 mM pH 6.8 phosphate buffer, wash with 1 mL buffer, dry under vacuum, elute with 1 mL MeOH water 40 60. Evaporate the eluate to dryness under a stream of nitrogen at 40°, reconstitute with 35 p-L MeOH water 5 95, iiyect a 25 pL aliquot. 

Internal standard thiosalicylic acid-p-bromophenacyl bromide adduct (Prepare by dissolving 2.4 mmoles thiosalicylic acid and 2.4 mmoles p-bromophenaQ l bromide in 40 mL MeOH, ac just to pH 7 by the dropwise addition of 1 M NaOH, allow to stand at room temperature for 10 min, evaporate to dryness under reduced pressure, reconstitute with 40 mL 50 mM pH 7.0 phosphate buffer, wash twice with 20 mL portions of hexane, adjust pH to 2 with dilute HCl, extract with 40 mL ethyl acetate, evaporate to dryness under reduced pressure, recrystallize the residue from benzene to give the adduct as pale yellow plates.)... 

Sample preparation Plasma. 1 mL Plasma -i- 2 mL 100 mM pH 6.0 phosphate buffer + 500 pL 0.5% N-(4-benzoylphenyl)maleimide in acetone, vortex for 15 s, let stand at room temperature for 10 min, add 2 mL 500 mM pH 7.0 phosphate buffer, add 100 pL 40 pg/mL ISl in acetone, wash twice with 4 mL portions of ether, acidify the aqueous phase with 500 pL 6 M HCl, extract with 7 mL chloroform. Remove the organic layer and evaporate it to dryness, reconstitute the residue in 100 pL MeOH, iiyect a 20 pL aliquot. Urine. 200 pL Urine + 200 pL 0.5% N-(4-benzoylphenyl)maleimide in acetone -I- 200 pL 100 mM pH 6.5 phosphate buffer, mix, let stand at room temperature for 15 min, add 2.5 mL 500 mM pH 7.0 phosphate buffer, wash with 4 mL diethyl ether, add 100 pL 10 pg/mL IS2 in acetone to the aqueous phase, acidify with 500 pL 6 M HCl, extract with 6 mL chloroform. Remove the organic layer and evaporate it to dryness, reconstitute the residue in 200 pL MeCN, inject a 20 pL aliquot. (Prepare N-(4-benzoylphenyl)maleimide by adding 5.3 g maleic anhydride to 9.6 g 4-aminobenzophenone in dioxane (Caution Dioxane is a carcinogen ), stir at room temperature (Japan Pat. 59,204,171 (19 Nov. 1984) Chem. Abstr. 1985, 102, 113288t).)... 

Sample preparation Condition a 3 mL 40 p,m bonded silica Clean Screen SPE cartridge (Worldwide Monitoring) with 3 mL MeOH, 3 mL water, and 1 mL pH 3 phosphate buffer. 1 mL Plasma + 2 mL 10 mM phosphoric acid, mix, add to the SPE cartridge, air dry for 30 s, wash with 1 mL pH 3 phosphate buffer, wash with 1 mL MeOH, air dry for 30 s, elute with 3 mL 2% ammoniacal MeOH. Evaporate the eluate to dryness under a stream of nitrogen at 45°, reconstitute the residue in 50 p,L mobile phase, inject an aliquot. 

A reactivation of an inactive nitrate reductase apoenzyme extracted from molybdenum-deficient plants can be achieved by the addition of acid-treated nitrate reductase or by addition of phosphate buffer washes of nitrate reductase absorbed on AMP-Sepharose. The acid treatment or phosphate wash apparently produced a molybdenum-containing complex which was responsible for reactivation of the apoenzyme. The complex had a molecular weight of less than 3 x 10 (Notton and Hewitt, 1971 Rucklidgee/< /., 1976). These reports clearly demonstrate the requirement for molybdenum for nitrate reduction however, the role of the metal in the reduction process is not completely resolved. 

Sample preparation Condition a 1 mL 100 mg Bond Elut C8 SPE cartridge with 1 mL MeOH and 1 mL 100 mM HCl. Dilute urine with an equal volume of water. Mix 1 mL plasma or diluted urine with 100 p,L IS in water and 1 mL 100 mM HCl, add to the SPE cartridge, wash with 2 mL 10 mM pH 7 phosphate buffer, wash with 500 m-L MeCNilO mM pH 7 phosphate buffer 20 80, elute with 2 mL MeCNilO mM pH 7 phosphate buffer 40 60, inject a 100 p.L aliquot of the eluate. 

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