What causes low yield and poor purity in protein purification from lysate?

[AubreyGR]

I'm trying to isolate a specific protein from a complex tissue lysate, but my yield is consistently low and the purity isn't what my protocol says it should be. I'm wondering if the issue is with my binding conditions during the affinity chromatography step, or if the sample preparation is degrading the target before it even gets to the column.

[Jerry.L]

I've had this exact vibe for months. Sometimes the protein just refuses to stay on the resin, even when the buffer matches the protocol. I keep chasing binding conditions—salt, pH, resin age—and the yield stays stubbornly low.

[StellaRM]

In my last run I started adding a broad protease inhibitor cocktail to the lysate and kept everything cold, and I did notice more of the protein staying on during binding, but the purity still wasn't great after elution.

[MichaelT]

I keep wondering if the kit or lysate prep is removing tags or something; the bands look different on the gel and I worry the target isn't intact.

[LarryH]

Maybe the real bottleneck isn't the column at all; the signal in the crude lysate could be overwhelmed, or the protein could be unstable and degrade during handling.