Phosphorylated proteins often have different conformations or changes in their mobility, which could be more pronounced or less distinguishable depending on the gel system. Bis-Tris gels might alter protein conformation in a way that changes the separation of phosphorylated versus non-phosphorylated forms.

Sometimes the conditions for Western blotting, such as transfer efficiency or the blocking and antibody conditions, may not be fully optimized for the Bis-Tris gel system. It's possible that the proteins or phosphorylated forms are not transferred as efficiently, or there could be an issue with the antibody recognition in the context of the Bis-Tris gel.

Testing these possibilities one by one should help pinpoint the cause of the problem :

Check the pH and ionic strength of the buffers used in your Bis-Tris gels. You could try adjusting the running buffer or the gel composition to see if it makes a difference.

Bis-Tris gels require different transfer protocols.

Try Different Antibodies or Blocking Agents: If you suspect the phosphorylation-specific antibody may not be optimal for this new gel system, you could try another antibody or change the blocking conditions to see if this improves the results.