Why do we use different PH in stacking gel and separating gel...

I performed SDS-PAGE by using total bacterial protein samples obtained by sonicaition method having volume of 40 micro litre and protein marker of 20 micro litre. But in this experiment I am not able to get the protein bands after destaining process. I like to know is what is possible trouble shooting in my experiment.

Laemmli-SDS-PAGE system is discontinuous buffer system. Different PH (and ionic strength) in stacking gel and separating gel can help protein samples concentrate/stack in stacking gel before running in a separating gel. Thus, the resolution of discontinuous buffer system much better than that of continuous systems (in particular when the volume of a protein sample is large).

Running buffer contains glycerine, not glycerol. Glycerine is a weak acid used to adjust the pH of the running buffer.

Glycerol in protein sample buffer is heavier (more dense) than water so that it makes protein sample sink at the bottom of the sample well instead of flowing around in the upper running buffer.