The appearance of separate heavy chain and light chain conjugate bands on your non-reducing SDS-PAGE is due to the TCEP reduction step, which irreversibly cleaves the interchain disulfide bonds of the antibody, generating free thiols that are then conjugated with the maleimide-containing drug. As a result, the antibody's subunits are no longer covalently linked, and when denatured with SDS, even without reducing agent, they migrate as individual chains during electrophoresis. In contrast, papers showing intact antibody-drug conjugate bands despite using a similar protocol likely experience partial reformation of disulfide bonds during handling or purification, often through unintended air oxidation. Even without an explicit re-oxidation step, exposure to air over time can allow disulfide bonds to reform, restoring interchain connectivity and resulting in a full antibody-sized band on SDS-PAGE. To replicate this, you can allow mild air oxidation after conjugation or introduce a gentle oxidizing agent like oxidized glutathione. Additionally, avoiding boiling and minimizing SDS during sample preparation may preserve enough structural integrity to visualize intact ADC bands.

Saif Ahmed Hussein Alabedi Thanks for your help, too, Saif.