Antibodies, Proteins, Kits and Reagents for Life Science

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100 µl 318 288 ₸ 10 µl 127 315 ₸

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

Key features and details

Produced recombinantly (animal-free) for high batch-to-batch consistency and long term security of supply
Rabbit monoclonal [EPR20018-251] to DDDDK tag (Binds to FLAG® tag sequence)
Suitable for: WB, ICC/IF, Flow Cyt, IHC-P, IP
Reacts with: Species independent

Product name

Anti-DDDDK tag (Binds to FLAG® tag sequence) antibody [EPR20018-251]
See all DDDDK tag (Binds to FLAG® tag sequence) primary antibodies
Description

Rabbit monoclonal [EPR20018-251] to DDDDK tag (Binds to FLAG® tag sequence)
Host species

Rabbit
Tested applications

Suitable for: WB, ICC/IF, Flow Cyt, IHC-P, IPmore details
Species reactivity

Reacts with: Species independent
Immunogen

Synthetic peptide. This information is proprietary to Abcam and/or its suppliers.
Positive control
- WB: HEK-293 transfected with PFKFB3 and HEK-293 transfected with PD-L1 cell lysate. IHC-P: HEK-293T transfected with DDDDK-tagged human PD-L1. ICC/IF: HEK-293T cells. Flow: HEK-293T transfected with DDDDK-tagged human PD-L1. IP: HEK-293T transfected with DDDDK-tagged human PFKFB3.
General notes
FLAG^® is a registered trade mark of Sigma Aldrich Biotechnology LP. It is used here for informational purposes only.
This product is a recombinant monoclonal antibody, which offers several advantages including:
- - High batch-to-batch consistency and reproducibility
- - Improved sensitivity and specificity
- - Long-term security of supply
- - Animal-free production
For more information see here.
Our RabMAb^® technology is a patented hybridoma-based technology for making rabbit monoclonal antibodies. For details on our patents, please refer to RabMAb^® patents.
The Life Science industry has been in the grips of a reproducibility crisis for a number of years. Abcam is leading the way in addressing the problem with our range of recombinant monoclonal antibodies and knockout edited cell lines for gold-standard validation.

One factor contributing to the crisis is the use of antibodies that are not suitable. This can lead to misleading results and the use of incorrect data informing project assumptions and direction. To help address this challenge, we have introduced an application and species grid on our primary antibody datasheets to make it easy to simplify identification of the right antibody for your needs.

Learn more here.

Form

Liquid
Storage instructions

Shipped at 4°C. Store at +4°C short term (1-2 weeks). Upon delivery aliquot. Store at -20°C long term. Avoid freeze / thaw cycle.
Storage buffer

pH: 7.2
Preservative: 0.01% Sodium azide
Constituents: PBS, 40% Glycerol (glycerin, glycerine), 0.05% BSA
Concentration information loading...
Purity

Protein A purified
Clonality

Monoclonal
Clone number

EPR20018-251
Isotype

IgG
Research areas

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - 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.
Immunocytochemistry/ Immunofluorescence - 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.
Western blot - Anti-DDDDK tag (Binds to FLAG® tag sequence) antibody [EPR20018-251] (ab205606)

All lanes : 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
Lane 2 : HEK-293 transfected with PFKFB3 (WT) expression vector containing an N-terminal DDDDK tag, whole cell lysate

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

Observed band size: 60 kDa
why is the actual band size different from the predicted?

Blocking/Diluting buffer: 5% NFDM/TBST

Exposure time: 0.5 seconds
Western blot - Anti-DDDDK tag (Binds to FLAG® tag sequence) antibody [EPR20018-251] (ab205606)

All lanes : 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
Lane 2 : HEK-293 transfected with PD-L1 (WT) expression vector containing a C- terminal DDDDK tag, whole cell lysate

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

Blocking/Diluting buffer: 5% NFDM/TBST

Exposure time: 1 second
Immunoprecipitation - Anti-DDDDK tag (Binds to FLAG® tag sequence) antibody [EPR20018-251] (ab205606)

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

All lanes : Anti-DDDDK tag (Binds to FLAG® tag sequence) antibody [EPR20018-251] (ab205606) at 1.2 µg/ml (1:500 dilution)

Lane 1 : HEK-293 (human epithelial cell line from embryonic kidney) whole cell lysate
Lane 2 : NIH/3T3 (mouse embryo fibroblast cell line) whole cell lysate
Lane 3 : C6 (rat glial tumor cell line) whole cell lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution (Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated)

Developed using the ECL technique.

Observed band size: 25,30,45,80 kDa why is the actual band size different from the predicted?

Exposure time: 20 seconds

Blocking/Diluting buffer and concentration: 5% NFDM/TBST.

ab205606 can detect extra bands in non-transfected cell lines at low dilution.
Flow Cytometry - Anti-DDDDK tag (Binds to FLAG® tag sequence) antibody [EPR20018-251] (ab205606)

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.
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-DDDDK tag (Binds to FLAG® tag sequence) antibody [EPR20018-251] (ab205606)

Negative control: No staining on rat spleen.

Immunohistochemical analysis of paraffin-embedded rat spleen tissue stained for DDDDK tag using ab205606 at 1/750 dilution, followed by a ready to use Goat Anti-Rabbit IgG H&L (HRP). Counterstained with hematoxylin.

Heat mediated antigen retrieval was performed using EDTA buffer pH 9 before commencing with IHC staining protocol.
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-DDDDK tag (Binds to FLAG® tag sequence) antibody [EPR20018-251] (ab205606)

Negative control: No staining on mouse spleen.

Immunohistochemical analysis of paraffin-embedded mouse spleen tissue stained for DDDDK tag using ab205606 at 1/750 dilution, followed by a ready to use Goat Anti-Rabbit IgG H&L (HRP). Counterstained with hematoxylin.

Heat mediated antigen retrieval was performed using EDTA buffer pH 9 before commencing with IHC staining protocol.
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-DDDDK tag (Binds to FLAG® tag sequence) antibody [EPR20018-251] (ab205606)

Negative control: No staining on human hepatocellular cancer.

Immunohistochemical analysis of paraffin-embedded human hepatocellular cancer tissue stained for DDDDK tag using ab205606 at 1/750 dilution, followed by a ready to use Goat Anti-Rabbit IgG H&L (HRP). Counterstained with hematoxylin.

Heat mediated antigen retrieval was performed using EDTA buffer pH 9 before commencing with IHC staining protocol.
Anti-DDDDK tag (Binds to FLAG® tag sequence) antibody [EPR20018-251] (ab205606)

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