Lysonase

Manufactured by Merck Group

Lysonase is a laboratory reagent used in the enzymatic lysis of bacterial cells. It contains the enzyme lysozyme, which breaks down the peptidoglycan layer of bacterial cell walls, facilitating the release of cellular contents for further analysis or processing.

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Lab products found in correlation

Problock gold bacterial 2d, by GoldBio (3 mentions) Pierce anti dykddddk magnetic agarose, by Thermo Fisher Scientific (1 mentions) Sypro ruby protein blot stain, by Thermo Fisher Scientific (1 mentions) Chemidoc mp system, by Bio-Rad (1 mentions)

Spelling variants of this product

BugBuster Plus Lysonase Kit (2 citations) Lysonase Bioprocessing Reagent (2 citations)

5 protocols using lysonase

Purification of His-tagged Proteins

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Cells were suspended in lysis buffer (50 mM HEPES, pH 7.5, 125 mM NaCl, 1 mM TCEP, 1X ProBlock Gold Bacterial 2D, Gold Bio) and lysed by the combined action of lysonase (Sigma Millipore) and ultrasonication. Cell-free extract was prepared by centrifugation at 29,500 g for 45 min at 4°C, and His₁₀-tagged proteins were purified using Pierce Anti-DYKDDDDK Magnetic Agarose (Thermo Fisher Scientific), according to the manufacturer’s protocol.

Hao Z., Epshtein V., Kim K.H., Proshkin S., Svetlov V., Kamarthapu V., Bharati B., Mironov A., Walz T, & Nudler E. (2020). Pre-Termination Transcription Complex: Structure and Function. Molecular cell, 81(2), 281-292.e8.

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Protein Purification by Lysis and Affinity

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Cells were suspended in lysis buffer (50 mM HEPES, pH 7.5, 500 mM NaCl, 2 mM MgSO₄, 5 mM ZnSO₄, 1 mM TCEP, 1X ProBlock Gold Bacterial 2D, Gold Bio) and lysed by the combined action of lysonase (Sigma Millipore) and ultrasonication. A cell-free extract was prepared by centrifugation at 29,500 g for 45 min at 4°C, and His₁₀-tagged proteins were purified using His Mag Sepharose Ni (GE Healthsciences), according to the manufacturer’s protocol.

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Purification of His-tagged Proteins

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The cells were suspended in the lysis buffer (50 mM HEPES (pH 7.5), 500 mM NaCl, 2 mM MgSO₄, 5 mM ZnSO₄, 1 mM TCEP, 1X ProBlock Gold Bacterial 2D, Gold Bio) and lysed by the combined action of Lysonase (Sigma Millipore) and ultrasonication. Cell-free extract was prepared by centrifugation at 4 °C, 29 500 g, 45 min, and His₁₀-tagged proteins were purified using His Mag Sepharose Ni (GE Healthsciences), according to manufacturer protocol.

Bharati B.K., Gowder M., Zheng F., Alzoubi K., Svetlov V., Kamarthapu V., Weaver J.W., Epshtein V., Vasilyev N., Shen L., Zhang Y, & Nudler E. (2022). Essential Role and Mechanism of Transcription-coupled DNA Repair in Bacteria. Nature, 604(7904), 152-159.

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Fluorinated Oil-Based Enzymatic Assay

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The materials used were purchased from Sigma Aldrich unless otherwise indicated. Fluorinated oils HFE-7500 were purchased from 3 M Novec and the 008-Fluorosurfactant from RAN biotechnologies. Resorufin-N-acetyl-β-D-galactosamine was purchased from Markergene (M1037), EDTA-free protease inhibitor from Roche (11873580001), Lysonase (71230), and BugBuster (70921-4) from Millipore.

Tauzin A.S., Pereira M.R., Van Vliet L.D., Colin P.Y., Laville E., Esque J., Laguerre S., Henrissat B., Terrapon N., Lombard V., Leclerc M., Doré J., Hollfelder F, & Potocki-Veronese G. (2020). Investigating host-microbiome interactions by droplet based microfluidics. Microbiome, 8, 141.

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Quantitative Protein Expression Analysis

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Strains harboring fusion genes or vector control were diluted to OD 0.05 in LB with IPTG (100 μM with msGFP2 fusions; 0, 50, or 500 μM with 3×FLAG fusions) and grown to OD 0.5. Cells were centrifuged and resuspended (50 μL per 1 mL of sample) with SDS sample loading buffer (msGFP2 fusions) or BugBuster protein extraction reagent with 0.1% Lysonase (Millipore) followed by SDS loading buffer (3×FLAG fusions). Samples were boiled for 10 min, separated by SDS-PAGE (12% acrylamide gel), and transferred to an Immobilon-FL polyvinylidene difluoride (PVDF) membrane. Total protein was detected by SYPRO ruby protein blot stain (Invitrogen). GFP was detected by rabbit PABG1 primary (Chromotek; 1:5,000 dilution) and goat anti-rabbit IgG horseradish peroxidase (HRP) secondary (Invitrogen; 1:5,000 dilution) antibodies. FLAG epitope was detected by mouse anti-FLAG M2 primary (Invitrogen; 1:1,000 dilution) and goat anti-mouse IgG HRP secondary (Invitrogen; 1:5,000 dilution) antibodies. Blots were imaged with a ChemiDoc MP system (Bio-Rad). Band intensities were quantified using Image Lab software. Samples were normalized by dividing the immunodetected band intensity by the total protein level from SYPRO staining. Relative values were calculated by dividing each normalized value by the normalized value of the ElsL_FLAG, 50 μM IPTG sample on the same blot.

Dai Y., Pinedo V., Tang A.Y., Cava F, & Geisinger E. (2021). A New Class of Cell Wall-Recycling l,d-Carboxypeptidase Determines β-Lactam Susceptibility and Morphogenesis in Acinetobacter baumannii. mBio, 12(6), e02786-21.

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