Freeze-thaw is not an efficient method of breaking the cells, although it may release some protein. You can also add EDTA to destabilize the outer membrane and lysozyme to destroy the peptidoglycan cell wall. Osmotic shock (e.g. distilled water) may then improve cell lysis. (see attached file)

Frothing during sonication should definitely be avoided. It does not cause the formation of inclusion bodies, but it does have the potential to denature the protein you are trying to isolate. Frothing can usually be prevented by keeping the power setting low enough and immersing the probe deeply enough. The sound you hear during sonication should be like that of food frying. If frothing occurs, stop immediately and let the air rise to the surface before continuing. Use multiple short bursts with cooling in between. Heating of the sample can be minimized by using a stainless steel beaker set in a mixture of ice and water, since steel is a much better heat conductor than glass or plastic.

If there is a French press available at your institution, it is a very effective way of breaking open E. coli cells for protein extraction without heating. I use two passes at 18,000 psi.

Another way to break the cells is with a bead beater, which is a relatively inexpensive piece of equipment. Preventing overheating of the sample is a problem with it, though.

There are also detergent-based reagents available that can be used to break open the cells, if you don't mind getting detergent in your sample. Because of the expense, the reagent is best used for small amounts of bacteria.

https://www.thermofisher.com/order/catalog/product/78243#:~:text=The%20B%2DPER%20Reagent%20solution,will%20leak%20out%20cytosolic%20proteins.

Dear Anu Jaglan

as suggeste bt Adam B Shapiro freeze thaw coupled with Lysozime addiction may work well in small scale. However it is difficulte to be scaled up. Sonication or cell press are preferable. Alternative for medium volumes you can use also chemical lyssi (eg detergent based compounds as Sigma Cell Lyitic Express or Thermo scientific B-per supplementad with lysozime)

At the following link

https://proteocool.blogspot.com/2021/08/tips-of-week11-chemicalenzymatic-cell.html

on my blog, ProteoCool ProteoCool

you can find a post:

Pills#11: Chemical_enzymatic cell lysis as alternative to physical lysis in high-throughput small scale protein purification from E.coli

the could be usefull to you!

best regards

Manuele