I am working on yeast display library screening.

The yeast library express nanobody on their cell wall together with HA-tag.

All steps were following the protocol described in published literature.

After 48-72h expression induction by 2% Galactose in selection media, I tested expression level with fluorophore-labeled-anti-HA-antibody. It was as low as 7%(in the literature, they recommend 15-20%).

Then I separated yeasts with MACS (from Miltenyi company), recover them, and induce again. This time, yeasts only have