Quantifying Epitope-Tagged Protein Expression

HEK-293T cells were transfected with viral gene vectors, each which encode in-frame 2x Strep tag fusion as described in [42 (link)]. Twenty-four hours post transfection, cells were collected and washed with PBS containing 0.5% BSA, before fixation with 2% PFA in PBS for 15 minutes at RT. After this, cells were washed twice, then permeabilized using ice cold methanol for 30 minutes at -20°C. Cells were then washed before incubation with anti-Strep antibody for 1h on ice, according to the manufacture’s protocol (IBA, strepMAB-Classic 2-1507-001), washed and incubated with biotinylated anti-mouse antibody for 1 hour on ice. Finally, cells were washed and incubated with Alexa Fluor 647 streptavidin (Jackson Immuno Research, AB_2341101) for 15 minutes on ice and were subjected to FACS analysis.