Hello,

I have been trying to optimize a western blot to detect CACNA1C using Alomone's CACNA1C ACC-003 antibody. Given the high molecular weight of the protein, I was advised to use Tris-Acetate gels.

• Gel: 3-8% gradient Tris-Acetate gel
• Sample Loading: 30 µg of protein per well (from lysed brain tissues)
• Lysis: Bioruptor system (new protocol for us), LP buffer + β-mercaptoethanol, 5-minute boil
• Gel Running Conditions: 150V for 1 hour
• Transfer: Wet transfer (100V for 30 min, then 50V for another 30 min)
• Blocking: 5% milk powder

Despite multiple runs, I consistently encounter the following issues:

The red in the first image shows the ladder and a well with sample for a different antibody that didnt seem to work. The green is the CACNA1C and the loading control.

I run a loading control (sodium-potassium ATPase), which shows a band but is blurred due to high background.

I’d appreciate any insights on troubleshooting these artifacts. Could this be an issue with the sample prep, transfer conditions, or something else? Has anyone successfully optimized conditions for high MW proteins like CACNA1C using Tris-Acetate gels?

Thanks in advance for any suggestions!