I’m doing cell-surface biotinylation with HEK293 cells and after all the washing and biotin incubation steps (washing with PBS ph8, neutralizing unreacted biotin with 40mM Tris ph8) many of my cells lift off. I do my washes extremely slowly and carefully to no avail. I have gotten new buffers, new biotin, new HEK cells all with passage numbers under 20. Some of my petris are fine but some lose almost all the cells and I end up getting very little protein. What should I do?