Team:Tec-Monterrey/projectprotocols

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Soluble and insoluble protein extraction from bacterial cell culture & Preparation of samples for SDS-PAGE

6 ml of transformed cell culture is harvested by centrifugation at 10 – 12,000 x g for 1 min.
The supernatant is removed and the pellet is dried.
For each 1 g of cell pellet, 20 ml of xTractor Buffer, 40 µl of DNAse 200 µl of 100X lysozyme solution is added.
The suspension is incubated during 10 min at room temperature.
The crude lysate is centrifugated at 10 – 12000 x g for 20 min and the supernatant is called as soluble fraction and the pellet as insouble fraction.
The insoluble fraction is sonicated with water during about 5 sec.
50 µl of protein fraction is mixed with 50 µl of 2x sample buffer with 2-ME and heat 10 min at 95 °C.