For the ChIRP MS experiment, I am sonicating the crosslinked HT29 cell lysate using diagenode bioruptor.

The cell lysis buffer has 50mM tris-Cl (pH7.0), 10mM EDTA (pH8.0), 1% SDS, 1mM PMSF, 1X PIC, and 200U RNase inhibitor.

I have tried multiple sonication conditions starting:

Pulse On - 5 secs, Pulse OFF -45 secs for 5 to 10 cycles,

Pulse ON - 15 secs, Pulse OFF - 45 secs for 5 to 10 cycles,

Pulse ON - 30 secs, Pulse OFF - 30 secs for 5-10 secs,

Pulse ON - 30 secs, Pulse OFF - 45 secs for 5 to 60 cycles.

After sonication, I am doing proteinase K treatment (50mM tris-Cl (pH7.0), 10mM EDTA (pH8.0), 100mM NaCl, 0.5% SDS and 5% PK enzyme).

I am also doing reverse crosslinking after proteinase K treatment, but still not observing smear bands.

I expect to observe a smara band above 1 kb. But it not showing in either of the sonication conditions. What can be the possible reason?

What is the lower prominent dark band in the gel at the bottom of each sample?

Every time my 1% agarose gel image has been like this: (I am sharing a sample gel image to look over) (P- pellet and S - supernatant separated during the proteinase K treatment).