Found 1 discussion for this experiment
2 years ago
2 years ago by Hollie Fowler
I am using 8M Urea to lyse my cells but after centrifugation my pellet is very viscous. This causes me trouble when I try to collect the supernatant. Is there any way to avoid it and do you think it will have an effect on the concentration of my protein?
Found 3 matching solutions for this experiment
|- Cultures are harvested by centrifugation (3000 g, 30 mins) and the cell pellet is washed in Buffer A (50 mM Tris pH 8.0, 500 mM KCl, 12.5 mM MgCl2, 1.5% 1,2 propanediol or ethanolamine-HCl).
|- Pellets are resuspended in 10 mL Buffer A and cells are lysed by addition of B-PER® II Bacterial Protein Extraction Reagent (60% v/v) (ThermoScientific).
- B-PER® II Bacterial Protein Extraction Reagent is supplemented with lysozyme (0.25 mg mL−1), DTT (1 mM), PMSF (0.1 mM) and DnaseI (2 U).
- After 30 minutes incubation at RT, cell debris and supernatant are separated twice by centrifugation (9700 g, 5 mins).
|- If you want to isolate bacterial microcompartments: BMCs are pelleted from the resulting supernatant by centrifugation (43000 g, 30 mins). The pellet is resuspended in 500–1000 μL Buffer B (50 mM Tris pH 8.0, 50 mM KCl, 5 mM MgCl2, 1% 1,2 propanediol or ethanolamine-HCl).|
|- For soluble protein preparations from up to 4.5 liters of bacterial cell culture.
- Before use, Native Lysis Buffer must be supplemented with lysozyme and Benzonase® Nuclease.
|- Downstream applications are SDS-PAGE and mass spectrometry.
- Solubilization of inclusion bodies requires high concentrations of denaturants (e.g., 6M Guanidine HCl).