Inefficient cell lysis may be occurring due to the following:
Kit solutions were improperly stored.
Keep the lysis and solubilization reagent at 4℃ at all times when not in use. The two binding buffers should be kept at room temperature.
Freeze/thaw step was not performed.
The freeze/thaw step is known to increase lysis efficiency. Repeat the protocol using the recommended freeze/thaw conditions.
Lysozyme may be required to increase lysis efficiency.
Add lysozyme to concentrations recommended by the supplier.
Mechanical disruption of cells was inefficient.
Increase the number of passages through the needle and syringe.