CelLytic™ B Cell Lysis Reagent

Protocol tips

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	- Cells are lysed by sonication in lysis buffer [400 mM NaCl, 20 mM Tris⋅HCl, pH 8.0, lysozyme 600 μg/mL, 4 μL of Benzonase Dnase (Sigma), 0.5 mL of CelLytic B (Sigma), 1 tablet of EDTA-free protease inhibitor mixture (cOmplete; Roche), 1% (vol/vol) Triton X-100].

Protocol tips
- Cells are lysed by sonication in lysis buffer [400 mM NaCl, 20 mM Tris⋅HCl, pH 8.0, lysozyme 600 μg/mL, 4 μL of Benzonase Dnase (Sigma), 0.5 mL of CelLytic B (Sigma), 1 tablet of EDTA-free protease inhibitor mixture (cOmplete; Roche), 1% (vol/vol) Triton X-100].

Publication protocol

Sheath-like structures were purified from both V. cholerae and E. coli host strains by using a two-step purification method. Protein expression was induced at OD600 = 0.5–0.7 by 1 mM IPTG and 0.2% arabinose (only for V. cholerae strains carrying the pTARA plasmid, to induce expression of T7 RNA polymerase) for 3 h. Cells were lysed by sonication in lysis buffer [400 mM NaCl, 20 mM Tris⋅HCl, pH 8.0, lysozyme 600 μg/mL, 4 μL of Benzonase Dnase (Sigma), 0.5 mL of CelLytic B (Sigma), 1 tablet of EDTA-free protease inhibitor mixture (cOmplete; Roche), 1% (vol/vol) Triton X-100]. Previously cleared lysates were subject to ultraspeed centrifugation at 150,000 × g for 1 h at 4 °C to concentrate the sample and remove soluble proteins not assembled as sheaths, as previously reported. In addition, the use of Triton X-100 in lysis buffer reduced the possibility to copurify membrane structures and outer membrane vesicles during the ultracentrifugation.

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Manufacturer protocol

Download the product protocol from Sigma-Aldrich for CelLytic™ B Cell Lysis Reagent below.

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