Common problems when ultrasonic homogenizer breaks Escherichia coli

E. coli expresses foreign protein. When sonicated, it is suspended in PBS containing 1% triton-X-100. The ultrasonic effect is better. The effect of 1% triton-X-100 is still obvious. Works, such as Streptomyces.

Bacterial precipitation directly add sample 1buffer, add 5ml of mercaptoethanol, mix, centrifuge, boil for 10min, directly load, staining and decoloring steps are as follows: put the glue into the appropriate amount of dyeing liquid in the microwave oven for 1min, the dyeing solution is replaced by a large amount The water can be cooked in the microwave for 10 minutes.

After expressing the recombinant protein, the cells were disrupted by ultrasonic wave, using an ice bath, 400W, and 2S stopped for 1S, but a large amount of foam was generated in a short time, which affected the crushing efficiency. Both pbs and tris buffers were the same, and finally the crushing was incomplete. But my target protein is in these unbroken cells, which makes me unable to get experimental results.

Let me give you an analysis of why this situation and solution.

1. Bubbles will be generated because your probe position is not placed well, the probe must be close to the bottom, about 1cm (I am usually 0.5cm from the bottom), the instrument will have different power levels, but you can observe the page, there are fluctuations but Don't be too aggressive.

2. Super 3S stops 10S, ultrasound about 20 times to see the effect

3. Pay attention to the position of the horn. If you are not correct, you should adjust it in time. In addition, from the perspective of the concentration of bacteria, try to increase the volume when crushing, the strength should not exceed 60%.

4. Try 8S for Super 8S. For some bacterial proteins, your method is difficult to dissipate heat, resulting in protein denaturation and bubble generation. It is best to pause for a little longer. This is more common in the form of inclusion bodies.