Anti-DDDDK tag (Binds to FLAG® tag sequence) antibody [EPR20018-251](AB205606)

Immunhistochemical analysis of agarose-embedded HEK-293T (Human epithelial cell line from embryonic kidney transformed with large T antigen) transfected with DDDDK-tagged human PD-L1 expression vector labeling DDDDK tag with ab205606 at 1/750 dilution, followed by a ready to use Goat Anti-Rabbit IgG H&L (HRP). Counterstained with hematoxylin.

Positive staining on HEK-293T cells transfected with DDDDK-tagged human PD-L1 expression vector (Panel A) is observed. No signal was detected on HEK-293T transfected with an empty vector (vector control), containing a C-terminal DDDDK tag (Panel B).

Heat mediated antigen retrieval was performed using EDTA buffer pH 9 before commencing with IHC staining protocol.

Flow cytometric analysis of of 4% paraformaldehyde-fixed, 90% methanol permeabilized HEK-293T (human epithelial cell line from embryonic kidney transformed with large T antigen) transfected with DDDDK-tagged human PD-L1 expression vector labeling DDDDK tag with ab205606 at 1/700 dilution (Red) compared with the Rabbit monoclonal IgG isotype control (ab172730) (Black) and an unlabeled control (cells without incubation with primary antibody and secondary antibody) (Blue). Goat anti rabbit IgG (Alexa Fluor® 488, ab150077), at 1/2000 dilution was used as the secondary antibody.

DDDDK tag was immunoprecipitated from 0.35 mg HEK-293T (human epithelial cell line from embryonic kidney transformed with large T antigen) transfected with DDDDK-tagged human PFKFB3 expression vector whole cell lysate with ab205606 at 1/30 dilution. Western blot was performed from the immunoprecipitate using ab205606 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/10000 dilution.

Lane 1: HEK-293T transfected with DDDDK-tagged human PFKFB3 expression vector whole cell lysate (input).

Lane 2: ab205606 IP in HEK-293T transfected with DDDDK-tagged human PFKFB3 expression vector whole cell lysate.

Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab205606 in HEK-293T transfected with DDDDK-tagged human PFKFB3 expression vector whole cell lysate.

Blocking/Dilution buffer: 5% NFDM/TBST

Exposure time: 3 seconds.

All lanes:

Immunoprecipitation - Anti-DDDDK tag (Binds to FLAG® tag sequence) antibody [EPR20018-251] (ab205606)

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized HEK-293T (Human epithelial cell line from embryonic kidney transformed with large T antigen) cells labeling DDDDK tag with ab205606 at 1/100 dilution, followed by ab150077 AlexaFluor®488 Goat anti-Rabbit secondary at 1/1000 dilution (green).

Confocal image showing positive staining for FLAG® on HEK-293T cells transfected with DDDDK-tagged PFKFB3 expression vector. Mouse monoclonal ANTI-FLAG® M2 antibody was used as a counterstain at 1/500 dilution, and AlexaFluor®647 Goat anti-mouse secondary (ab150115) was used as the secondary antibody only control at 1/500 dilution. The nucleus is counterstained with DAPI.

Blocking/Diluting buffer: 5% NFDM/TBST

Exposure time: 0.5 seconds

All lanes:

Western blot - Anti-DDDDK tag (Binds to FLAG® tag sequence) antibody [EPR20018-251] (ab205606) at 1/1000 dilution

Lane 1:

HEK-293 (human epithelial cell line from embryonic kidney) transfected with an empty vector (vector control), containing an N-terminal DDDDK tag, whole cell lysate at 10 µg

Lane 2:

HEK-293 transfected with PFKFB3 (WT) expression vector containing an N-terminal DDDDK tag, whole cell lysate at 10 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

Observed band size: 60 kDa

Blocking/Diluting buffer: 5% NFDM/TBST

Exposure time: 1 second

All lanes:

Western blot - Anti-DDDDK tag (Binds to FLAG® tag sequence) antibody [EPR20018-251] (ab205606) at 1/5000 dilution

Lane 1:

HEK-293 (human epithelial cell line from embryonic kidney) transfected with an empty vector (vector control), containing a C-terminal DDDDK tag, whole cell lysate at 10 µg

Lane 2:

HEK-293 transfected with PD-L1 (WT) expression vector containing a C- terminal DDDDK tag, whole cell lysate at 10 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution