Western blot analysis of DYKDDDDK Tag (FLAG) on DYKDDDDK Tag recombinant protein (80kDa). Proteins were transferred to a PVDF membrane and blocked with 5% BSA in PBS for 1 hour at room temperature. The primary antibody (0912-1, 1/5,000) was used in 5% BSA at room temperature for 2 hours. Goat Anti-Rabbit IgG - HRP Secondary Antibody (HA1001) at 1:5,000 dilution was used for 1 hour at room temperature.

Western blot analysis of DYKDDDDK Tag (FLAG) on different lysates. Proteins were transferred to a PVDF membrane and blocked with 5% BSA in PBS for 1 hour at room temperature. The primary antibody (0912-1, 1/2,000) was used in 5% BSA at room temperature for 2 hours. Goat Anti-Rabbit IgG - HRP Secondary Antibody (HA1001) at 1:5,000 dilution was used for 1 hour at room temperature.
Lane 1: N-terminal FLAG-tagged recombinant protein
Lane 2: C-terminal FLAG-tagged recombinant protein

DYKDDDDK Tag (FLAG) was immunoprecipitated from 0.2 mg L-929 transfected with FLAG-tagged CD5 cell lysate with 0912-1 at 2 µg/10 µl beads. Western blot was performed from the immunoprecipitate using 0912-1 at 1/1,000 dilution. HRP Conjugated Anti-Rabbit IgG for IP Nano-secondary antibody at 1/5,000 dilution was used for 1 hour at room temperature.

Lane 1: L-929 transfected with FLAG-tagged CD5 cell lysate (input)
Lane 2: 0912-1 IP in L-929 transfected with FLAG-tagged CD5 cell lysate
Lane 3: Rabbit IgG instead of 0912-1 in L-929 transfected with FLAG-tagged CD5 cell lysate

Blocking/Dilution buffer: 5% NFDM/TBST
Exposure time: 3 seconds; ECL: K1801