Product Class: Other

NEBExpress® T4 Lysozyme
cloned at NEB recombinant


Catalog #P8115

Product Introduction

  • NEBExpress T4 Lysozyme is 200-fold more active than chicken egg white lysozyme.
  • Lysis reactions are scalable and compatible with high throughput workflows.
  • Fast and non-mechanical bacterial lysis; the lysate is ready to use and compatible with affinity resins. 
  • 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.

Product Information

Description

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.

P8115 Figure 1

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.

P8115 Figure 2

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.

 
This product is related to the following categories:
Competent Cells Products,
E. coli Protein Expression Strains Products,
Protein Expression Products,
This product can be used in the following applications:
Protein Expression in E. Coli,
Protein Expression

Reagents Supplied

Reagents Supplied

The following reagents are supplied with this product:

NEB # Component Name Component # Stored at (°C) Amount Concentration

Properties & Usage

Storage Buffer

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

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.
 

Product Notes

  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. 

References

  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.

Protocols, Manuals & Usage

Protocols

  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 Spin gDNA Extraction Kit (NEB #T3010)
  3. Protocol for Genomic DNA Extraction from Gram-positive Bacteria using NEBExpress® T4 Lysozyme (NEB #P8115) and the Monarch Spin gDNA Extraction Kit (NEB #T3010)

FAQs & Troubleshooting

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)?
  11. Is NEBExpress T4 Lysozyme tagged?

Troubleshooting

Quality, Safety & Legal

Quality Assurance Statement

Quality Control tests are performed on each new lot of NEB product to meet the specifications designated for it. Specifications and individual lot data from the tests that are performed for this particular product can be found and downloaded on the Product Specification Sheet, Certificate of Analysis, data card or product manual. Further information regarding NEB product quality can be found here.

Certificate Of Analysis

The Certificate of Analysis (COA) is a signed document that includes the storage temperature, expiration date and quality controls for an individual lot. The following file naming structure is used to name these document files: [Product Number]_[Size]_[Version]_[Lot Number]

Safety DataSheets

The following is a list of Safety Data Sheet (SDS) that apply to this product to help you use it safely.

Legal and Disclaimers

Products and content are covered by one or more patents, trademarks and/or copyrights owned or controlled by New England Biolabs, Inc (NEB). The use of trademark symbols does not necessarily indicate that the name is trademarked in the country where it is being read; it indicates where the content was originally developed. The use of this product may require the buyer to obtain additional third-party intellectual property rights for certain applications. For more information, please email busdev@neb.com.

This product is intended for research purposes only. This product is not intended to be used for therapeutic or diagnostic purposes in humans or animals.

New England Biolabs (NEB) is committed to practicing ethical science – we believe it is our job as researchers to ask the important questions that when answered will help preserve our quality of life and the world that we live in. However, this research should always be done in safe and ethical manner. Learn more.

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