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

Problem Possible causes Solution
No clearance of the lysate after incubation. Cell suspension is too dense. Lack of clearance does not mean the cells did not lyse well.  Add 10-20% v/v more Lysis Reagent to the lysate and incubate at room temperature for 5-10 minutes.
Lysate is too viscous. DNA release causes the lysate to become viscous, which affects collection/processing of the supernatant. Add 200 - 2000 U/mL of Micrococcal Nuclease (NEB #M0247) or 10 to 100 U/mL DNase I (NEB #M0303) with 1 mM CalCl2 final to the viscous lysate, mix, and incubate at room temperature for 5 minutes, or until the viscosity decreases, before the centrifugation step.
Protein of interest (POI) is insoluble. The POI may form inclusion bodies. Optimize expression conditions to improve POI solubility (change expression strain, induction, or growth conditions). For example, expressing at a lower temperature and/or with less inducer may improve solubility. If using an E. coli expression system, solubility can be improved by choosing a strain that:
  • Tightly controls basal expression of the target gene with lacIq or lysY such as T7 Express lysY/Iq (NEB #C3013), T7 Express lysY (NEB #C3010) or NEBExpress lacIq (NEB #C3037)
  • Is designed for tunable T7 expression of the target such as Lemo21(DE3) (NEB #C2528)
  • Allows disulfide bond formation in the cytoplasm (SHuffle® T7 Express - NEB #C3029)
Visit our webpages for more information about strains and expression strategies:
Solubilize inclusion bodies recovered in the insoluble fraction by following common denaturation and refolding strategies.
No or low amount of protein of interest (POI) detected by SDS-PAGE. Lysis could be incomplete. Resuspend cells in not less than 10 µL Lysis Reagent per UOD600 of cells or 6 mL per gram of wet cells and add 1 µL of NEBExpress® T4 Lysozyme (NEB #P8115) per 1 mL of lysate with 200 - 2000 U/mL of Micrococcal Nuclease (NEB #M0247) and 1 mM CaCl2 to improve lysis.
Amount of protein loaded on gel could be too low. Load at least 10-20 µL of supernatant mixed with 5-10 µL of Blue Protein Loading Dye (NEB #B7703S).
POI expression level might be too low. Western blot or activity assays could be used to detect the POI.

Improve expression conditions to harvest more POI: refer to the protocol of the strain used or visit our webpages for more information on E. coli strains and protein expression strategies:
POI is degraded. Avoid vortexing the lysate and try adding protease inhibitors to help reduce protein degradation.