I'm currently performing EMSA (gel shift) experiments to examine protein-DNA interactions. Specifically, I am testing whether the transcription factor TFEB binds to the CLEAR motif in the presence of APOE. My cells are cultured with an APOE knockdown, and I intend to compare binding with and without APOE expression.

In my recent experiments, I used the following conditions:

• Gel: Native polyacrylamide Tris-Glycine gel
• Running Buffer: Tris-Glycine native running buffer (without SDS)
• Transfer Buffer: Tris-Glycine buffer without methanol

my results did not show any clear shift, indicating possible issues with complex formation or transfer efficiency. I also observed relatively poor resolution and transfer efficiency.

My questions are:

the reason I didn't use recommended TBE buffer system is because it involves in radiation and I don't have that access.