Immunocytochemistry analysis of HeLa cells labeling DYKDDDDK Tag (FLAG) with Mouse anti-DYKDDDDK Tag (FLAG) antibody (HA610017) at 1/250 dilution. HeLa cells, transfected with FLAG-tagged empty control, Claudin 18.2 (C-terminal) or Histone H3.1 (N-terminal) expression vector, respectively, were fixed in 4% paraformaldehyde for 10 minutes at room temperature, permeabilized with 0.1% Triton X-100 in PBS for 15 minutes at room temperature, then blocked with 1% BSA in 10% negative goat serum for 1 hour at room temperature. Cells were then incubated with Mouse anti-DYKDDDDK Tag (FLAG) antibody (HA610017) at 1/250 dilution in 1% BSA in PBST overnight at 4 ℃. Goat Anti-Mouse IgG H&L (iFluor™ 488, HA1125) was used as the secondary antibody at 1/1,000 dilution. PBS instead of the primary antibody was used as the secondary antibody only control. Nuclear DNA was labelled in blue with DAPI. beta Tubulin (ET1602-4, red) was stained at 1/100 dilution overnight at +4℃. Goat Anti-Rabbit IgG H&L (iFluor™ 594, HA1122) were used as the secondary antibody at 1/1,000 dilution.

FLAG-tag was immunoprecipitated in 2µg C-terminal FLAG-tag fusion protein lysate with HA610017. Western blot was performed from the immunoprecipitate using HA610017 at 1/5,000 dilution. Anti-Mouse IgG for IP Nano-secondary antibody (NBI02H) at 1:2,000 dilution was used for 60 mins at room temperature. Lane 1: C-terminal FLAG-tag fusion protein lysate (input). Lane 2: HA610017 IP in C-terminal FLAG-tag fusion protein lysate. Lane 3: Mouse IgG instead of HA610017 IP in C-terminal FLAG-tag fusion protein lysate. Blocking/Dilution buffer: 5% NFDM/TBST.