Just to clarify your question: on the gel, do you see nothing at all, or the lysate samples look smeared? do you load a ladder? how do you stain your gel, standard Coomassie protocol?

Also, since Staphylococcus aureus is a gram-positive bacterium, it is harder to break its cell wall as compared to E. coli. Which protocol do you use for cell lysis? Do you observe genomic DNA release during the procedure (which may serve as an indicator that the lysis was successful)?

I agreed with Victor G Stepanov discussions. I want to add one thing is that, If the protein is not produced by any plasmid vector (either under constitutive or inducible promoter), then you may also try a larger volume of culture for cell harvesting to ensure a sufficient amount of production.

Lysostaphin enzyme is recommended for lysis of S. aureus.

You could also just dissolve the cell pellet in SDS-PAGE sample buffer with heating, then centrifuge to remove undissolved material.

Victor G Stepanov Dear Victor, thanks a lot for the discussion. First of all, I do see bands I wouldn't say smeared but they are pale and not clear of course compared with the ladder. The main point for doing the SDS page is to make sure I can see enough proteins prior to analysis on LCMSMS.

The ladder is all standards blue. for the staining, after running the gel, I wash it three times with water after heating 1 minute after I use Coomassie blue, I heat it for 45 seconds and leave it overnight on the shaker.

For the cell lysis, First I dissolved the pellet in a lysate buffer which contains(SDS4%, TRIS-HCL, TRITON, DTT), followed by three times thermomixer and vortex for 5 minutes, then freeze-thaw three times, followed by Tissue laser, Sonication, and vortex 3 times, centrifugation, and the supernatant was added to chloroform, methanol, and water, centrifugation and precipitation, adding Laemmle, boiling then loading on the gel.

I tried different buffers and one time without freeze-thaw and tissue laser but every time, unfortunately, the bands don't show well. Do you recommend some changes?

It seems like you are trying to use only physical methods to disrupt the cells, is it because you do not want to introduce an external enzyme into your LCMSMS samples? It may not be sufficient in the case of Staphylococcus aureus, which is known for its sturdy cell wall. You might try using Lysostaphin in combination with sonication, something like 300 micrograms of the enzyme per approx. 10^9 TE-washed cells resuspended in 300 mcl of TE buffer with 0.3 M NaCl, 30 min at 37 oC. Then sonicate it on ice for 10 min, and remove insolubles by centrifugation. Supernatant should be viscous because of the released gDNA. The later can be removed by DNase treatment.

If you do not want to spike your samples with external enzymes, you may try exploiting autolysis of freshly-harvested S. aureus cells by resuspending them in 50 mM Sodium Phosphate buffer (pH 6.5) with 0.3 M NaCl, and incubating for 2 hours at 30 oC. Then sonicate the suspension and centrifigate it as above. This method relies on NaCl-induced autolysis of S. aureus cells with their own enzymes. It is somewhat more cumbersome as compared with Lysostaphin treatment.

People often use bead-beating to disrupt S. aureus cells but I have never tried it so cannot say anything about it.