Why is my protein purification yield so low with affinity chromatography?

[MilaKM]

Every platform has its podcast rankings and charts, but how much do you actually rely on them for finding new shows? Do these podcast rankings reflect quality, popularity, or just marketing budgets? I find myself increasingly skeptical of top charts and wondering if there are better ways to assess podcast quality than download numbers.

[Anthony.W]

I'm trying to isolate a specific protein from a complex tissue lysate, but my yield after the affinity chromatography step is consistently lower than what the literature suggests I should be getting. I'm worried my elution buffer's pH might be causing partial denaturation or that the binding capacity of the resin is being compromised somehow.

[Thomas_T]

I’ve seen this after the resin had a few dozen runs. At first the binding looked fine, but later the eluate was weaker and the target band on a quick gel was smaller.

[Emma2]

The pH thing matters, but so does salt, buffer components, and even small shifts that don’t denature the protein can change how tightly it sticks to the resin.

[Addison.W]

Make sure the tag is still accessible in your lysate; proteases can trim tags or the protein can adopt a conformation that hides the binding epitope.

[ScarlettYM]

Old or improperly regenerated resin can lose binding sites or carry residues that mess with elution; we’ve swapped resin and seen a jump in yield.

[Robert.C]

Sometimes the problem is in the lysate prep itself; too many particulates or high viscosity can keep the protein from hitting the resin efficiently.

[Brian_H]

Do you have a control with a known amount of tagged protein run through the same column to compare the elution profile?