DYKDDDDK Tag (FLAG) Recombinant Mouse Monoclonal Antibody [PS00-M2]

Western blot analysis of DYKDDDDK Tag (FLAG) on different lysates with Mouse anti-DYKDDDDK Tag (FLAG) antibody (HA601080) at different dilutions.

Lane 1/2/3: C-terminal FLAG-tag fusion protein lysate
Lane 4/5/6: N-terminal FLAG-tag fusion protein lysate

Lysates/proteins at 50 ng/Lane.

Observed band size: 35/36 kDa

Exposure time: 2 minutes;

12% SDS-PAGE gel.

Proteins were transferred to a PVDF membrane and blocked with 5% NFDM/TBST for 1 hour at room temperature. The primary antibody (HA601080) at different dilutions was used in 5% NFDM/TBST at room temperature for 2 hours. Goat Anti-Mouse IgG - HRP Secondary Antibody (HA1006) at 1:150,000 dilution was used for 1 hour at room temperature.

Immunocytochemistry analysis of HeLa cells labeling DYKDDDDK Tag (FLAG) with Mouse anti-DYKDDDDK Tag (FLAG) antibody (HA601080) 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 (HA601080) 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 HA601080. Western blot was performed from the immunoprecipitate using HA601080 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: HA601080 IP in C-terminal FLAG-tag fusion protein lysate.
Lane 3: Mouse IgG instead of HA601080 IP in C-terminal FLAG-tag fusion protein lysate.

Blocking/Dilution buffer: 5% NFDM/TBST.

FLAG-tag was immunoprecipitated in 2µg N-terminal FLAG-tag fusion protein lysate with HA601080. Western blot was performed from the immunoprecipitate using HA601080 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: N-terminal FLAG-tag fusion protein lysate (input).
Lane 2: HA601080 IP in N-terminal FLAG-tag fusion protein lysate.
Lane 3: Mouse IgG instead of HA601080 IP in N-terminal FLAG-tag fusion protein lysate.

Blocking/Dilution buffer: 5% NFDM/TBST.