|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:
|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.