New England Biolabs, P8115L, NEBExpress® T4 Lysozyme

Related Categories Competent Cells,, E. coli Protein Expression Strains,, Protein Expression Applications Protein Expression in, E. coli, ,, Protein Expression Specification 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 FAQ Q: How are NEBExpress® T4 Lysozyme and chicken egg white lysozyme different? A: NEBExpress® T4 Lysozyme is a recombinant protein ~200 fold more active than chicken egg white lysozyme when used under the same conditions. Therefore, much less T4 Lysozyme is needed to lyse E. coli in Tris buffer; ~1 μg of T4 lysozyme enzyme per 1 mL of E.coli cells versus ~0.1 to 0.5 mg of chicken egg white lysozyme per 1 mL of E.coli cells. The enzyme NEBExpress® T4 Lysozyme is 18.7 kDa, while chicken egg white lysozyme is 14.3 kDa. Q: Can I use NEBExpress® T4 Lysozyme for DNA or RNA extraction? A: Yes. NEBExpress® T4 Lysozyme has no detectable endonuclease, exonuclease, RNase, or DNase activity and is therefore recommended for DNA and RNA extraction. Q: What buffers are compatible with NEBExpress® T4 Lysozyme? A: NEBExpress® T4 Lysozyme is compatible with Tris-HCl, HEPES, and sodium phosphate-based buffers. It has an optimal pH range of 7.5-8.5 and can be supplemented with <25 mM NaCl, <2 mM EDTA, <1 mM MgCl2, and Tween 20 or Triton X-100 <2%. The enzyme is also compatible with reducing agents such as DTT, β-mercaptoethanol, TCEP, and THP. For optimal T4 Lysozyme activity, use either: 1 µL NEBExpress T4 Lysozyme (1 µg of enzyme) per 1 mL of resuspended cells in 50 mM Tris-HCl pH 7.5, 0.5 mM EDTA. 0.1 µL NEBExpress T4 Lysozyme per 1 mL of resuspended cells in NEBExpress® E. coli lysis reagent (NEB #P8116S). Note that ≥ 2mM EDTA, > 25 mM NaCl, > 1 mM MgCl2 will start inhibiting enzyme activity, with a 50% loss of activity at 5 mM EDTA, 80% loss at 50 mM NaCl and 80% loss at 5 mM MgCl2. Q: Can reducing agents be used when lysing with NEBExpress® T4 Lysozyme? A: Yes, NEBExpress® T4 Lysozyme is compatible with commonly used reducing reagents, including β-mercaptoethanol, DTT, TCEP, and THP. For downstream purification, unlike other vendors, NEBExpress® Nickel Spin Columns (NEB #S1427) and NEBExpress® Nickel Resin (NEB #S1428) are chemically resistant to these reducing reagents. Q: Is it ok to dilute NEBExpress® T4 Lysozyme before adding it to my resuspended cells? A: Yes, If a very small volume of NEBExpress® T4 Lysozyme needs to be used, it can be diluted in 50 mM Tris-HCl pH 7.5. Q: What are the optimal incubation temperature and time for lysing cells with NEBExpress® T4 Lysozyme? A: Cells should be lysed with T4 Lysozyme at room temperature or 4˚C for 5-10 minutes with gentle shaking. Incubation at 37˚C is not recommended to avoid potential induction of protease activity. Q: Is NEBExpress®T4 Lysozyme active on organisms other than E. coli? A: Yes, NEBExpress® T4 Lysozyme can efficiently lyse other gram-negative bacteria such as Vibrio natriegens, Salmonella typhimurium, Pseudomonas putida, Xanthomonas malvacearum, Ralstonia eutropha, Flavobacterium johnsoniae as well as all E. coli expression strains available in the NEB catalog. It is also active on some gram-positive bacteria such as certain Bacillus strains (e.g., B. subtilis, B. thuringiensis). Note that 5 to 15-fold more NEBExpress T4 Lysozyme may be required to achieve lysis with a non-E. coli strain. Q: After incubation with NEBExpress® T4 Lysozyme, the lysate became viscous; how can I resolve this? A: A viscous lysate results from an abundance of DNA released. To reduce viscosity, three options are available: Shear the DNA by subjecting the lysate to a couple of pulses on a sonicator. Add 1mM CaCl2 and 1 µL of Micrococcal Nuclease (NEB #M0247S) per 1 mL of lysate, mix, and incubate for 5 minutes at room temperature. Add 1 µL of DNase I (NEB #M0303S) per 1 mL of lysate and 1 mM CaCl2 or 1 mM MgCl2 (especially if the suspension contains EDTA), mix, and incubate for 5 minutes at room temperature. NEB’s DNase I is tagged with maltose-binding protein (MBP), making its removal via amylose resin (NEB #E8021), amylose resin high flow (NEB #E8022), or amylose magnetic beads (NEB #E8035) simple and effective. Q: Should cell pellets be frozen prior to lysing with NEBExpress® T4 Lysozyme? A: Freezing is unnecessary; however, frozen cell pellets may increase lysis efficiency. Q: Can I use NEBExpress® T4 Lysozyme in a standard lysozyme activity assay using a fluorescent substrate (4-methylumbelliferone)? A: No. NEBExpress T4 Lysozyme hydrolyzes only the polysaccharide substrate, composed of a chain of N-acetylglucosamine (GlcNAc) and N-acetylmuramic acid (MurNAc) linked in β-1 → 4, when crosslinked with a pentapeptide chain. The linear polysaccharide substrate without the peptides is not a substrate for NEBExpress T4 Lysozyme. Q: Is NEBExpress T4 Lysozyme tagged? A: No. This enzyme is not tagged. However, due to its high pI (9.576), it is easily removed by cation exchange resin.