I’m currently running a Western blot to detect pSTAT3 (86 kDa) using a 10% resolving gel and a 4% stacking gel. Here's a summary of my protocol:

• Cell Lysis: I lyse 2 million cells using 45 µL RIPA buffer, followed by sonication (50% amplitude for 5 seconds). Then, I spin the lysate at 10,000 rpm for 10 minutes at 4°C.
• Sample Preparation: I add sample buffer with B-ME and incubate in a dry water bath for 5 minutes.
• Gel Running: I load 10 µL of the sample and run the gel at 120V for 2 hours in the cold room.
• Transfer: I transfer the proteins onto a membrane at 80V for 2 hours.
• Blocking & Antibody Incubation: I block the membrane with 5% skim milk for 1 hour, followed by primary antibody incubation overnight at 4°C. I incubate with the secondary antibody for 1 hour before detecting using ECL.

One additional detail is that I cut the membrane after transfer.

Despite following this protocol, I’m not getting a strong signal for pSTAT3. Could anyone offer troubleshooting tips or suggestions to improve my results?