D-alanine esterified to teichoic acids was detected and quantified as described by27 (link). Briefly, D-alanine was released from whole heat-inactivated bacteria by mild alkaline hydrolysis with 0.1 N NaOH for 1h at 37°C. After neutralization, the extract was incubated with Marfey's reagent (1-fluoro-2,4-dinitrophenyl-5-L-alanine amide; Sigma). This reagent reacts with the optical isomers of amino acids to form diastereomeric N-aryl derivatives, which can be separated by HPLC. Separation of the amino acid derivatives was performed on a C18 reversed-phase column (Zorbax Eclipse Plus C18 RRHD 2.1x50mm 1.8µm Agilent) with an Agilent UHPLC 1290 system with a linear elution gradient of acetonitrile in 20 mM sodium acetate buffer (pH 4) as described previously 27 (link). The eluted compounds were detected by UV absorbance at 340 nm. Quantification was achieved by comparison with D-alanine standards in the range of 100 to 1500 pmol. Mean values were obtained from five independent cultures with two injections for each.
Zorbax eclipse plus c18 rrhd 2.1x50mm 1 8 m
Manufactured by Agilent Technologies
The Zorbax Eclipse Plus C18 RRHD 2.1x50mm 1.8µm is a high-performance liquid chromatography (HPLC) column designed for rapid and efficient separation of a wide range of analytes. The column features a 1.8 μm porous silica particle size and a C18 stationary phase, providing high-resolution chromatographic performance.
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2 protocols using zorbax eclipse plus c18 rrhd 2.1x50mm 1 8 m
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Quantification of D-Alanine in Bacteria
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Quantification of D-Alanine in Bacteria
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D-alanine esterified to teichoic acids was detected and quantified as described by27 (link). Briefly, D-alanine was released from whole heat-inactivated bacteria by mild alkaline hydrolysis with 0.1 N NaOH for 1h at 37°C. After neutralization, the extract was incubated with Marfey's reagent (1-fluoro-2,4-dinitrophenyl-5-L-alanine amide; Sigma). This reagent reacts with the optical isomers of amino acids to form diastereomeric N-aryl derivatives, which can be separated by HPLC. Separation of the amino acid derivatives was performed on a C18 reversed-phase column (Zorbax Eclipse Plus C18 RRHD 2.1x50mm 1.8µm Agilent) with an Agilent UHPLC 1290 system with a linear elution gradient of acetonitrile in 20 mM sodium acetate buffer (pH 4) as described previously 27 (link). The eluted compounds were detected by UV absorbance at 340 nm. Quantification was achieved by comparison with D-alanine standards in the range of 100 to 1500 pmol. Mean values were obtained from five independent cultures with two injections for each.
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