My goal is to express my target protein using sf9 cells and I expect to see two bands because of the different post-translational modifications. I use western blot to check protein expression and I have been struggling to get the expected protein bands.

In my pilot purification, I successfully observed the desired ovulin bands such as fig1. However, during subsequent attempts to scale up the purification, I encountered difficulties that multiple bands appeared on the blot (fig2 and fig3). This issue persisted despite several different attempts. I suspected that the virus might be degraded or mutated. In an effort to troubleshoot the problem, I remade fresh bacmid DNA and promptly transfected the sf9 cells, attempting to replicate the conditions that yielded successful results. Unfortunately, the observed band pattern on the blot did not align with my expectations again. I am currently at a loss as to what might be causing this issue or how I can optimize the process.

Has anyone ever encountered or heard such situations? Are there any suggestions? Thanks very much!!