Biological replicates were grown to mid-logarithmic or stationary phase in 400 mL of Luria broth. Cells were centrifuged (Avanti JXN-26 Beckman Coulter centrifuge and Beckman Coulter JA-10 rotor) at 7,000 × g for 0.5 h at 4°C, resuspended in chilled 6 mL PBS, and lysed via dropwise addition to boiling 8% sodium dodecyl sulfate (SDS). PG was further purified as previously described (84 (link)). Briefly, muropeptides were cleaved from PG by Cellosyl muramidase (Hoechst, Frankfurt am Main, Germany), reduced with sodium borohydride, and separated on a 250- by 4.6-mm 3-μm Prontosil 120-3-C18 AQ reversed-phase column (Bischoff, Leonberg, Germany). The eluted muropeptides were detected by the absorbance at 205 nm. Eluted peaks were designated based on known published chromatograms (20 (link), 42 (link), 57 (link)); new peaks were analyzed by MS/MS, as previously described (20 (link)).
Jxn 26 centrifuge
Manufactured by Beckman Coulter
The JXN-26 Beckman Coulter centrifuge is a high-performance benchtop centrifuge designed for a wide range of laboratory applications. It features a maximum speed of 26,000 rpm and a maximum RCF of 54,600 x g, making it suitable for separating a variety of sample types. The centrifuge comes with a selection of interchangeable rotors to accommodate different tube sizes and configurations.
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2 protocols using jxn 26 centrifuge
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Purification and Analysis of Bacterial Peptidoglycan
2
Purification and Analysis of Bacterial Peptidoglycan
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Biological replicates were grown to mid-logarithmic or stationary phase in 400 mL of Luria broth. Cells were centrifuged (Avanti JXN-26 Beckman Coulter Centrifuge, Beckman
Coulter JA-10 rotor) at 7,000 x g for 0.5 h at 4C, resuspended in chilled 6 mL PBS and lysed via drop-wise addition to boiling 8% sodium dodecyl sulfate (SDS). PG was further purified as previously described 80 . Briefly, muropeptides were cleaved from PG by Cellosyl muramidase (Hoechst, Frankfurt am Main, Germany), reduced with sodium borohydride and separated on a 250-by 4.6-mm 3-μm Prontosil 120-3-C18 AQ reversedphase column (Bischoff, Leonberg, Germany). The eluted muropeptides were detected by absorbance at 205 nm. Eluted peaks were designated based on known published chromatograms 37, (link)44, (link)55 ; new peaks analyzed by MS/MS, as previously done 44 (link) .
Coulter JA-10 rotor) at 7,000 x g for 0.5 h at 4C, resuspended in chilled 6 mL PBS and lysed via drop-wise addition to boiling 8% sodium dodecyl sulfate (SDS). PG was further purified as previously described 80 . Briefly, muropeptides were cleaved from PG by Cellosyl muramidase (Hoechst, Frankfurt am Main, Germany), reduced with sodium borohydride and separated on a 250-by 4.6-mm 3-μm Prontosil 120-3-C18 AQ reversedphase column (Bischoff, Leonberg, Germany). The eluted muropeptides were detected by absorbance at 205 nm. Eluted peaks were designated based on known published chromatograms 37, (link)44, (link)55 ; new peaks analyzed by MS/MS, as previously done 44 (link) .
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