Express® T4 Lysozyme |NEB酶试剂 New England Biolabs

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产品信息

NEBExpress® T4 Lysozyme is a recombinant murein hydrolase that breaks down the bacterial cell wall by hydrolyzing the β-1,4 linkage between N-acetylmuramic acid and N-acetylglucosamine in the peptidoglycan of prokaryotic cells (gram-negative and some gram-positive bacteria).  It can be used to extract soluble proteins, membrane proteins, DNA, RNA, or metabolites. 

Source: An E. coli strain that carries the bacteriophage T4 gene e.

Figure 1: NEBExpress T4 Lysozyme is 200-fold more active than chicken egg white lysozyme.

Express® T4 Lysozyme |

T7 Express E. coli  expressing vGFP was lysed with NEBExpress T4 Lysozyme (T4L) or Chicken egg white lysozyme (ChL): 4 UOD600 of cell pellet were resuspended in 200 µL of 50 mM Tris-HCl pH 7.5 and lysed for 5 minutes at room temperature in the presence of NEBExpress T4 Lysozyme or Chicken egg white lysozyme at 0.1, 1, 10, 100 or 200 ug enzyme per 1 mL of cell suspension. The soluble proteins were harvested by centrifugation and analyzed on SDS-PAGE. 

Figure 2: Optimal protein extraction with NEBExpress T4 Lysozyme in the presence of NEBExpress E. coli Lysis Reagent.

Express® T4 Lysozyme |

T7 Express E. coli  expressing vGFP was lysed with NEBExpress T4 Lysozyme (T4L) in 50 mM Tris-HCl pH 7.5 or in NEBExpress E. coli Lysis Reagent (NEB #P8116S): 4 UOD600 of cell pellet were resuspended in 200 µL of Tris buffer or NEBExpress E. coli Lysis Reagent and lysed for 5 minutes at room temperature in the presence of NEBExpress T4 Lysozyme at 0, 0.1 or 1 ug per 1 mL of cell suspension. The soluble proteins were harvested by centrifugation and analyzed on SDS-PAGE.

 
产品类别:
Competent Cells Products,
E. coli Protein Expression Strains Products,
Protein Expression Products

应用:
Protein Expression in E. Coli,
Protein Expression

  • 产品组分信息

    本产品提供以下试剂或组分：

    NEB # 名称 组分货号 储存温度 数量 浓度
    • P8115S     -20    
        NEBExpress® T4 Lysozyme P8115SVIAL -20 1 x 200 µg 1 mg/ml
    • P8115L     -20    
        NEBExpress® T4 Lysozyme P8115SVIAL -20 5 x 200 µg 1 mg/ml

  • 特性和用法

    贮存溶液

    50 mM Tris-HCl
    100 mM NaCl
    1 mM DTT
    0.1 mM EDTA
    0.03% Poloxamer 188
    50% Glycerol
    pH 7.5 @ 25°C

  • 优势和特性

    Features

    • NEBExpress T4 Lysozyme is 200-fold more active than chicken egg white lysozyme.
    • Lysis reactions are scalable and compatible with high throughput workflows.
    • Lysis efficiency increases 2-fold when used in combination with NEBExpress E. coli Lysis Reagent (NEB #P8116S).
    • Fast and non-mechanical bacterial lysis; the lysate is ready to use and compatible with affinity resins.
    • Suitable for lysing thermosensitive proteins vulnerable to mechanical lysis procedures
    • Compatible with downstream applications such as SDS-PAGE, Western blot, activity assays, immunoprecipitation, and downstream purification 
    • Small (200 µg) and large (5 x 200 µg) formats are suitable for lysing 7 or 35 grams of cells, respectively.
    • Recombinant and animal free.
     

  • 相关产品

    相关产品

    • P8116 NEBExpress E coli Lysis Reagent
    • 微球菌核酸酶
    • m0303-dnase-i-rnase-free
    • BL21(DE3) E. coli 感受态细胞
    • T7 表达 E. coli 感受态细胞（高效级）
    • Amylose Resin
    • 几丁质树脂
    • NEBExpress® Ni Spin Columns
    • NEBExpress® Ni Resin
    • Monarch® 基因组 DNA 提取试剂盒
    • T3060 Monarch HMW DNA Extraction Kit for Tissue

  • 注意事项

    1. NEBExpress T4 Lysozyme is compatible with: 

      • Tris, HEPES and sodium phosphate buffers ranging from pH 6.5 – 9.5; optimal pH is 7.5 – 8.5. 
      • 0.5 – 2 mM EDTA, which will improve lysis efficiency on gram negative bacteria. EDTA concentrations above 2mM start inhibiting enzyme activity, with a 50% loss at 5 mM EDTA.  
      • ≤ 25 mM NaCl; enzymatic activity is inhibited by 80% at 50 mM NaCl 
      • ≤ 1 mM MgCl2; enzymatic activity is inhibited by 80% at 5 mM MgCl2 
      • ≤ 2% Triton X-100 or Tween 20
      • Protease inhibitor cocktails (with ≤ 2 mM EDTA) 
      • Reducing agent such as DTT, β-mercaptoethanol, TCEP and THP. 
      • For optimal lysis performance, include NEBExpress E. coli Lysis Reagent (NEB #P8116S)
    2. NEBExpress T4 Lysozyme can be added directly to resuspended cells or pre-mixed with the buffer prior to resuspension of the cell pellet.
    3. Use 1 µL of NEBExpress T4 Lysozyme per 1 mL of cells resuspended at 10 UOD600. For example, for a 5 mL culture harvested at OD600 of 2, 10 UOD600 of cells are obtained (5 mL x 2 OD600 = 10 UOD600). 
    4. If weighing the cell pellet, use 30 µL of NEBExpress T4 Lysozyme per 1 gram of cell paste resuspended in 30 mL buffer. 
    5. The lysate may become viscous due to the release of DNA. For optimal lysis yield, eliminate viscosity with Micrococcal Nuclease (NEB #M0247).  Add 1mM CaCl2 and 1 uL of Micrococcal Nuclease per 1 mL of lysate, mix and incubate for 5 minutes at room temperature.

    6. Lysis efficiency can be strain dependent. For hard to lyse samples, use 5 to 15 µL of NEBExpress T4 Lysozyme per 1 mL of resuspended cells.

    7. Cell pellets subjected to one freeze-thaw cycle prior to NEBExpress T4 Lysozyme treatment tend to lyse better. 

  • 参考文献

    1. Jensen, H.B., Kleppe K. (1972). Effect of Ionic Strength, pH, Amines and Divalent Cations on the Lytic Activity of T4 Lysozym. Eur. J. Biochem. 28, 116-122.

操作说明、说明书 & 用法

  • 操作说明

    1. NEBExpress® T4 Lysozyme Lysis Protocol (NEB #P8115)
    2. Protocol for Genomic DNA Extraction from Gram-negative Bacteria using NEBExpress® T4 Lysozyme (NEB #P8115) and the Monarch® Genomic DNA Purification Kit (NEB #T3010)
    3. Protocol for Genomic DNA Extraction from Gram-positive Bacteria using NEBExpress® T4 Lysozyme (NEB #P8115) and the Monarch® Genomic DNA Purification Kit (NEB #T3010)

FAQs & 问题解决指南

  • FAQs

    1. How are NEBExpress® T4 Lysozyme and chicken egg white lysozyme different?
    2. Can I use NEBExpress® T4 Lysozyme for DNA or RNA extraction?
    3. What buffers are compatible with NEBExpress® T4 Lysozyme?
    4. Can reducing agents be used when lysing with NEBExpress® T4 Lysozyme?
    5. Is it ok to dilute NEBExpress® T4 Lysozyme before adding it to my resuspended cells?
    6. What are the optimal incubation temperature and time for lysing cells with NEBExpress® T4 Lysozyme?
    7. Is NEBExpress®T4 Lysozyme active on organisms other than E. coli?
    8. After incubation with NEBExpress® T4 Lysozyme, the lysate became viscous; how can I resolve this?
    9. Should cell pellets be frozen prior to lysing with NEBExpress® T4 Lysozyme?
    10. Can I use NEBExpress® T4 Lysozyme in a standard lysozyme activity assay using a fluorescent substrate (4-methylumbelliferone)?

  • 问题解决指南

    • Troubleshooting Guide for NEBExpress E. coli Lysis Reagent (NEB # P8116)