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 2 matching solutions for this experiment
|- Bacterial pellets are recovered after centrifugation (10,000rpm, 10 min), washed twice in PBS, and resuspended in 100 µl of B-PER II bacterial protein extraction reagent following the manufacturer's protocol.|
|- Culture supernatants are precipitated in 60% ammonium sulfate or trichloroacetic acid at 4°C overnight. After centrifugation, pellets are washed twice with 5ml cold acetone, dried, and resuspended in 200 µl of 2× Laemmli buffer.|
|- Cultures are centrifuged (10,000×g, 10 min, 4°C), pellets are resuspended in 500µL of PBS and centrifuged (14,000×g, 5 min, 4°C). The PBS wash is repeated twice.
- Cell pellets are frozen using liquid nitrogen then thawed on ice for ≈15 min.
|- Soluble proteins were extracted from the cell pellets using a Qproteome bacterial protein preparation kit according to the manufacturer's protocol..|