What can fix low protein yield in affinity chromatography?

[Matthew_G]

I'm trying to isolate a specific protein from a complex tissue lysate for functional analysis, but my yield is consistently too low for the downstream assays. I think the issue might be happening during the affinity chromatography step, as my elution fractions show a lot of non-specific binding even after optimizing the wash buffer's ionic strength.

[OliviaXH]

I know the feeling. We chased cleaner fractions for weeks and kept seeing extra proteins in the elution even after more washes. It starts to feel like the resin is not selective enough for your target.

[Sofia_G]

We did switch the resin chemistry for the purification step and that helped the specificity a bit, but the yield took a hit. We kept the wash ionic strength high and tried a shorter binding phase but the eluted mix still showed noise. With affinity chromatography we saw new issues too.

[MiaUD]

Maybe the real bottleneck is the lysate quality or the tag accessibility rather than the step itself. If the target is degraded or hidden by partners, you end up chasing non specific stuff.

[Zoey_W]

One day we even ran a quick desalting pass to remove salts and that shifted the elution profile in a strange way. It felt like a distraction from the core problem but it did change the numbers, briefly.