I am doing a GST-tag protein purification. I have already an optimized protocol and it worked before. I am taking samples from before and after binding the lysate to beads and incubate 1.5 hours to bind to beads in cold room (called lysate and flow through respectively). So, recently I started to see no bands, like completely empty lane in flow-through. What could be the reason? I changed all buffers and they are all fresh. I boiled all samples at the same time. I am using PMSF as protease inhibitor.

I am adding an image. Lanes are: pellet, lysate, flow through, elution, beads