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2, the expression of recombinant protein, bacterial lysis methods are what?

When expressed recombinant proteins, induction and subsequent SDS-PAGE found that expression was good, but encountered problems in lysis of cells. Always can not completely lyse cells.

First of all, I use ultrasonic pyrolysis, but no matter how I ultrasound, there is always some bacteria did not lyse.

My ultrasound conditions are as follows: Ningbo Shinchi ultrasonic cell crusher, power 400W, over 5 seconds, 5 seconds apart, repeat 99 times. Then microscopic observation, incomplete again when repeated 99 times. Cells from 200 ml of culture medium, with 20 ml PBS resuspended.

Then I try to add lysozyme, first added to a final concentration of 100 ug / ml, 4C semi-hour bacteria solution does not become sticky, increasing the concentration to 1 mg / ml, still no significant change after half an hour. Because I use PBS is pH7.4, I suspect that the pH is not suitable for use with pH8.0 TE buffer, 1 mg / ml lysozyme, 4C half an hour still no significant sticky. Because the lysozyme used this day is a good day before, worried about the failure of lysozyme, lyophilized powder re-weighed directly into the bacterial liquid, still no significant change.

Really depressed. What happened? Please master solutions, and introduce the optimization program.

At the same time hope to introduce how to choose the method of cell disruption, and how to determine the best conditions.

Is lysozyme inactive at pH 7.4? Can be equipped with a concentrated solution of lysozyme, if so, how to save?

After adding lysozyme, if the cell wall is damaged, the bacteria should become sticky because the nucleic acid is released.

And ultrasound broken cells, I think the bacteria will not become sticky, because the ultrasound can break the macromolecular nucleic acid into small pieces.

My judgment of incomplete cell disruption is because, after the disrupted sample was centrifuged to separate the supernatant from the pellet and run on SDS-PAGE, it was observed that in addition to the frequently occurring bands or bands in the cell debris fraction, A lot of bands appear. According to this can be judged that the cells are only partially broken.