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Anti-DDDDK tag (Binds to FLAG® tag sequence) antibody [EPR20018-251] is a rabbit monoclonal antibody that is used in DDDDK epitope tag western blot (WB), immunoprecipitation (IP), flow cytometry, immunohistochemistry on paraffin-embedded sections (IHC-P), and immunocytochemistry/immunofluorescence (ICC/IF). Suitable for FLAG® tag samples.

- Cited in over 115 publications
- Recombinant format for unrivaled batch-batch consistency: no need for same-lot requests
- Specificity and sensitivity confirmed in IHC with multi-tissue microarray (TMA) validation
- Affinity purified using Protein A

Same trusted quality, new lower price


Images

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-DDDDK tag (Binds to FLAG® tag sequence) antibody [EPR20018-251] (AB205606), expandable thumbnail
  • Flow Cytometry - Anti-DDDDK tag (Binds to FLAG® tag sequence) antibody [EPR20018-251] (AB205606), expandable thumbnail
  • Immunoprecipitation - Anti-DDDDK tag (Binds to FLAG® tag sequence) antibody [EPR20018-251] (AB205606), expandable thumbnail
  • Immunocytochemistry/ Immunofluorescence - Anti-DDDDK tag (Binds to FLAG® tag sequence) antibody [EPR20018-251] (AB205606), expandable thumbnail
  • Western blot - Anti-DDDDK tag (Binds to FLAG® tag sequence) antibody [EPR20018-251] (AB205606), expandable thumbnail

Publications

Key facts

Isotype

IgG

Host species

Rabbit

Storage buffer

pH: 7.2 - 7.4
Preservative: 0.01% Sodium azide
Constituents: PBS, 40% Glycerol (glycerin, glycerine), 0.05% BSA

Form

Liquid

Clonality

Monoclonal

Immunogen

  • The exact immunogen used to generate this antibody is proprietary information.

Reactivity data

Select an application
Product promiseTestedExpectedPredictedNot recommended
IPFlow CytWBIHC-PICC/IF
Tag
Tested
Tested
Tested
Tested
Tested

Tested
Tested

Species

Tag

Dilution info

1/30

Notes

-

Tested
Tested

Species

Tag

Dilution info

1/700

Notes

-

Tested
Tested

Species

Tag

Dilution info

1/500 - 1/5000

Notes

This antibody can be used at 1.2 μg/ml. ab205606 can detect extra bands in non-transfected cell lines at low dilution.

Tested
Tested

Species

Tag

Dilution info

1/750

Notes

Perform heat-mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

Tested
Tested

Species

Tag

Dilution info

1/100

Notes

-

Associated Products

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Alternative names

Recommended products

Anti-DDDDK tag (Binds to FLAG® tag sequence) antibody [EPR20018-251] is a rabbit monoclonal antibody that is used in DDDDK epitope tag western blot (WB), immunoprecipitation (IP), flow cytometry, immunohistochemistry on paraffin-embedded sections (IHC-P), and immunocytochemistry/immunofluorescence (ICC/IF). Suitable for FLAG® tag samples.

- Cited in over 115 publications
- Recombinant format for unrivaled batch-batch consistency: no need for same-lot requests
- Specificity and sensitivity confirmed in IHC with multi-tissue microarray (TMA) validation
- Affinity purified using Protein A

Same trusted quality, new lower price

Key facts

Isotype

IgG

Form

Liquid

Clonality

Monoclonal

Immunogen
  • The exact immunogen used to generate this antibody is proprietary information.
Clone number

EPR20018-251

Purification technique

Affinity purification Protein A

Concentration
Loading...

Storage

Shipped at conditions

Blue Ice

Appropriate short-term storage duration

1-2 weeks

Appropriate short-term storage conditions

+4°C

Appropriate long-term storage conditions

-20°C

Aliquoting information

Upon delivery aliquot

Storage information

Avoid freeze / thaw cycle

Notes

Our RabMAb® technology is a patented hybridoma-based technology for making rabbit monoclonal antibodies. For details on our patents, please refer to RabMAb® patents.

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 batch production

For more information, read more on recombinant antibodies.

FLAG® is a registered trade mark of Sigma Aldrich Biotechnology LP. It is used here for informational purposes only.

Product promise

We are dedicated to supporting your work with high quality reagents and we are here for you every step of the way should you need us.

In the unlikely event of one of our products not working as expected, you are covered by our product promise.

Full details and terms and conditions can be found here:
Terms & Conditions.

24 product images

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

    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.

  • Flow Cytometry - Anti-DDDDK tag (Binds to FLAG® tag sequence) antibody [EPR20018-251] (ab205606), expandable thumbnail

    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 (Rabbit IgG, monoclonal [EPR25A] - 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, Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) ab150077), at 1/2000 dilution was used as the secondary antibody.

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

    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) (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 (Rabbit IgG, monoclonal [EPR25A] - Isotype Control 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)

  • Immunocytochemistry/ Immunofluorescence - Anti-DDDDK tag (Binds to FLAG® tag sequence) antibody [EPR20018-251] (ab205606), expandable thumbnail

    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 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) 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 (Goat Anti-Mouse IgG H&L (Alexa Fluor® 647) 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), expandable thumbnail

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

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

    Observed band size: 60 kDa

  • Western blot - Anti-DDDDK tag (Binds to FLAG® tag sequence) antibody [EPR20018-251] (ab205606), expandable thumbnail

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

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

  • Western blot - Anti-DDDDK tag (Binds to FLAG® tag sequence) antibody [EPR20018-251] (ab205606), expandable thumbnail

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

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

    ab205606 can detect extra bands in non-transfected cell lines at low dilution.

    All lanes: Western blot - Anti-DDDDK tag (Binds to FLAG® tag sequence) antibody [EPR20018-251] (ab205606) at 1.2 µg/mL

    Lane 1: HEK-293 (human epithelial cell line from embryonic kidney) whole cell lysate at 20 µg

    Lane 2: NIH/3T3 (mouse embryo fibroblast cell line) whole cell lysate at 20 µg

    Lane 3: C6 (rat glial tumor cell line) whole cell lysate at 20 µg

    Secondary

    All lanes: Western blot - Goat Anti-Rabbit IgG H&L (HRP) (Goat Anti-Rabbit IgG H&L (HRP) ab97051) at 1/2000 dilution

    Developed using the ECL technique.

    Observed band size: 25 kDa, 30 kDa, 45 kDa, 80 kDa

    Exposure time: 20s

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

    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), expandable thumbnail

    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), expandable thumbnail

    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.

  • Western blot - Anti-DDDDK tag (Binds to FLAG® tag sequence) antibody [EPR20018-251] (ab205606), expandable thumbnail

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

    Blocking buffer and concentration: 5% NFDM/TBST 

    Diluting buffer and concentration: 5% NFDM/TBST

    Exposure Time: Lane 1-2: 1 second, Lane 3: 40 seconds.

    Anti-GAPDH antibody [EPR16891] - Loading Control ab181602 was used as loading controls. ab205606 was used as a total protein control.

    Tag constructed at C -terminal of overexpression vector may interfere the recognition of this antibody to targeted protein.

    All lanes: Western blot - Anti-KLHL6 antibody [EPR13484] (Anti-KLHL6 antibody [EPR13484] ab182163) at 1/1000 dilution

    Lane 1: 293T (Human embryonic kidney epithelial cell) transfected with an empty vector (vector control) whole cell lysate at 20 µg

    Lane 2: 293T transfected with KLHL6 expression vector containing a N-terminal DDDDK tag, whole cell lysate at 20 µg

    Lane 3: Human tonsil lysate at 20 µg

    Secondary

    All lanes: Goat Anti-Rabbit IgG (HRP) with minimal cross-reactivity with human IgG at 1/2000 dilution

    Predicted band size: 70 kDa

    Observed band size: 70 kDa

  • Western blot - Anti-DDDDK tag (Binds to FLAG® tag sequence) antibody [EPR20018-251] (ab205606), expandable thumbnail

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

    Blocking/Dilution buffer: 5% NFDM/TBST

    Lane 1: Western blot - Anti-ABCG1 antibody [EP1366Y] (Anti-ABCG1 antibody [EP1366Y] ab52617) at 1/1000 dilution

    Lane 2: Western blot - Anti-DDDDK tag (Binds to FLAG® tag sequence) antibody [EPR20018-251] (ab205606) at 1/1000 dilution

    All lanes: C-Myc/DDK-tagged full length human ABCG1 recombinant protein at 10 ng

    Secondary

    All lanes: Western blot - Goat Anti-Rabbit IgG H&L (HRP) (Goat Anti-Rabbit IgG H&L (HRP) ab97051) at 1/20000 dilution

    Exposure time: 180s

  • Western blot - Anti-DDDDK tag (Binds to FLAG® tag sequence) antibody [EPR20018-251] (ab205606), expandable thumbnail

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

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

    This antibody does not cross-react with human MAP2 and MAP4.

    The antibody binds in the region surrounding Leu243 of 2N4R Tau

    In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control (Anti-GAPDH antibody [EPR16891] - Loading Control ab181602) staining at 1/200000 dilution.

    In Western blot, Anti-6X His tag® antibody [EPR20547] - ChIP Grade (Anti-6X His tag® antibody [EPR20547] - ChIP Grade ab213204) staining at 1/5000 dilution.

    In Western blot, Anti-DDDDK tag (Binds to FLAG® tag sequence) antibody(ab205606) staining at 37-250KDa dilution.

    All lanes: Western blot - Anti-Tau (MTBR-tau243) antibody [EPR29541-59] (Anti-Tau (MTBR-tau243) antibody [EPR29541-59] ab322554) at 1/1000 dilution

    Lane 1: 293T cells transfected with an empty vector containing a myc-His-tag®, whole cell lysate at 20 µg

    Lane 2: 293T cells transfected with a human wild-type Tau 2N4R expression vector containing a myc-His-tag®, whole cell lysate at 20 µg

    Lane 3: 293T cells transfected with a human wild-type PNS-Tau expression vector containing a DDDDK-tag, whole cell lysate at 20 µg

    Lane 4: 293T cells transfected with a human wild-type MAP2 expression vector containing a DDDDK-tag, whole cell lysate at 20 µg

    Lane 5: 293T cells transfected with a human wild-type MAP4 expression vector containing a DDDDK-tag, whole cell lysate at 20 µg

    Secondary

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

    Observed band size: 33-100 kDa, 36 kDa, 15-150 kDa, 37-250 kDa

    Exposure time: 6s

  • Western blot - Anti-DDDDK tag (Binds to FLAG® tag sequence) antibody [EPR20018-251] (ab205606), expandable thumbnail

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

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

    Exposure time: Lane 1, 2, 4, 5 , 6: 1 second. Lane 3: 80 seconds.

    Lanes 1 - 3: Western blot - Anti-IRS1+IRS2 antibody [EP263Y] (Anti-IRS1+IRS2 antibody [EP263Y] ab40777) at 1/1000 dilution

    Lanes 4 - 6: Western blot - Anti-DDDDK tag (Binds to FLAG® tag sequence) antibody [EPR20018-251] (ab205606) at 1/1000 dilution

    Lanes 1 and 4: HEK-293T transfected with an empty vector whole cell lysate at 20 µg

    Lanes 2 and 5: HEK-293T transfected with a human IRS1 expression vector containing a Myc/DDK tag whole cell lysate at 20 µg

    Lanes 3 and 6: HEK-293T transfected with a human IRS2 expression vector containing a Myc/DDK tag whole cell lysate at 20 µg

    Secondary

    All lanes: Western blot - Goat Anti-Rabbit IgG H&L (HRP) (Goat Anti-Rabbit IgG H&L (HRP) ab97051) at 1/20000 dilution

    Observed band size: 170 kDa

  • Western blot - Anti-DDDDK tag (Binds to FLAG® tag sequence) antibody [EPR20018-251] (ab205606), expandable thumbnail

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

    Blocking and diluting buffer: 5% NFDM/TBST.

    Anti-GAPDH antibody [EPR16891] - Loading Control ab181602 was used as a loading control at 1/1000000 dilution.

    ab205606 was used as a loading control at 1/1000 dilution.

    This antibody shows very weak cross reactivity with ACSL5 and ACSL6.

    All lanes: Western blot - Anti-ACSL1 antibody [EPR13498] (Anti-ACSL1 antibody [EPR13498] ab178419) at 1/1000 dilution

    Lane 1: 293T (human embryonic kidney epithelial cell) transfected with an empty vector whole cell lysate at 10 µg

    Lane 2: 293T transfected with a human ACSL1 expression vector containing a DDDDK tag whole cell lysate at 10 µg

    Lane 3: 293T transfected with a human ACSL5 expression vector containing a DDDDK tag whole cell lysate

    Lane 4: 293T transfected with a human ACSL6 expression vector containing a DDDDK tag whole cell lysate at 10 µg

    Secondary

    All lanes: Western blot - Goat Anti-Rabbit IgG H&L (HRP) (Goat Anti-Rabbit IgG H&L (HRP) ab97051) at 1/20000 dilution

    Predicted band size: 78 kDa

    Observed band size: 75 kDa

    Exposure time: 20s

  • Western blot - Anti-DDDDK tag (Binds to FLAG® tag sequence) antibody [EPR20018-251] (ab205606), expandable thumbnail

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

    Blocking and diluting buffer and concentration: 5% NFDM /TBST

    Anti-GAPDH antibody [EPR16891] - Loading Control ab181602 was used as a GAPDH loading control at 1/1000000 dilution.

    ab205606 was used at 1/1000 dilution. 

    All lanes: Western blot - Anti-ACSL1 + ACSL6 antibody [EPR13499] (Anti-ACSL1 + ACSL6 antibody [EPR13499] ab177958) at 1/1000 dilution

    Lane 1: 293T (human embryonic kidney epithelial cell) transfected with an empty vector whole cell lysate at 10 µg

    Lane 2: 293T transfected with a human ACSL1 expression vector containing a DDDDK tag whole cell lysate at 10 µg

    Lane 3: 293T transfected with a human ACSL5 expression vector containing a DDDDK tag whole cell lysate at 10 µg

    Lane 4: 293T transfected with a human ACSL6 expression vector containing a DDDDK tag whole cell lysate at 10 µg

    Secondary

    All lanes: Western blot - Goat Anti-Rabbit IgG H&L (HRP) (Goat Anti-Rabbit IgG H&L (HRP) ab97051) at 1/200000 dilution

    Observed band size: 75 kDa

    Exposure time: 3s

  • Western blot - Anti-DDDDK tag (Binds to FLAG® tag sequence) antibody [EPR20018-251] (ab205606), expandable thumbnail

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

    Blocking and dilution buffer: 5% NFDM/TBST.

    Lanes 1 - 2: Western blot - Anti-NAT10 antibody [EPR18663] (Anti-NAT10 antibody [EPR18663] ab194297) at 1/1000 dilution

    Lanes 3 - 4: Commercial benchmark Anti-NAT10 antibody

    Lanes 5 - 6: Western blot - Anti-DDDDK tag (Binds to FLAG® tag sequence) antibody [EPR20018-251] (ab205606) at 1/1000 dilution

    Lanes 1, 3 and 5: HEK-293T transfected with an empty vector whole cell lysate at 20 µg

    Lanes 2, 4 and 6: HEK-293T transfected with a human NAT10 expression vector containing a DDDDK tag whole cell lysate at 20 µg

    Secondary

    All lanes: Western blot - Goat Anti-Rabbit IgG H&L (HRP) (Goat Anti-Rabbit IgG H&L (HRP) ab97051) at 1/20000 dilution

    Predicted band size: 115 kDa

    Observed band size: 116 kDa

    Exposure time: 3s

  • Western blot - Anti-DDDDK tag (Binds to FLAG® tag sequence) antibody [EPR20018-251] (ab205606), expandable thumbnail

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

    Blocking and diluting buffer: 5% NFDM/TBST.

    ab205606 was used as a loading control at 1/1000 dilution.

    Anti-IMP3 antibody [EPR12021-114] ab179807 was used at 1/1000 dilution.

    Lanes 1 - 3: Western blot - Anti-IGF2BP2/IMP-2 antibody [EPR6741(B)] (Anti-IGF2BP2/IMP-2 antibody [EPR6741(B)] ab124930) at 1/1000 dilution

    Lanes 1 - 3: Western blot - Anti-IMP3 antibody [EPR12021-114] (Anti-IMP3 antibody [EPR12021-114] ab179807) at 1/1000 dilution

    Lane 1: C-Myc/DDK-tagged full length Human IGF2BP1 recombinant protein at 0.01 µg

    Lane 2: C-Myc/DDK-tagged full length Human IGF2BP2 recombinant protein at 0.01 µg

    Lane 3: N-His-tagged full length Human IGF2BP3 recombinant protein at 0.01 µg

    Secondary

    All lanes: Western blot - Goat Anti-Rabbit IgG H&L (HRP) (Goat Anti-Rabbit IgG H&L (HRP) ab97051) at 1/20000 dilution

    Predicted band size: 66 kDa

    Observed band size: 75 kDa

    Exposure time: 180s

  • Western blot - Anti-DDDDK tag (Binds to FLAG® tag sequence) antibody [EPR20018-251] (ab205606), expandable thumbnail

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

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

    ab205606 DDDDK tag was used as a loading control at 1/1000 dilution.

    All lanes: Western blot - Anti-IMP3 antibody [EPR12021-114] (Anti-IMP3 antibody [EPR12021-114] ab179807) at 1/1000 dilution

    Lane 1: C-Myc/DDK -tagged full length Human IGF2BP1 recombinant protein at 0.01 µg

    Lane 2: C-Myc/DDK -tagged full length Human IGF2BP2 recombinant protein at 0.01 µg

    Lane 3: N-His -tagged full length Human IGF2BP3 recombinant protein at 0.01 µg

    Secondary

    All lanes: Western blot - Goat Anti-Rabbit IgG H&L (HRP) (Goat Anti-Rabbit IgG H&L (HRP) ab97051) at 1/20000 dilution

    Predicted band size: 63 kDa

    Observed band size: 75 kDa

    Exposure time: 180s

  • Western blot - Anti-DDDDK tag (Binds to FLAG® tag sequence) antibody [EPR20018-251] (ab205606), expandable thumbnail

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

    Blocking and diluting buffer: 5% NFDM/TBST.

    This antibody shows weak cross reactivity with IGF2BP2.

    ab205606 was used as a loading control at 1/1000 dilution.

    Anti-IMP3 antibody [EPR12021-114] ab179807 was used at 1/1000 dilution.

    Lanes 1 - 3: Western blot - Anti-IGF2BP1/IMP1 antibody [EPR18791] (Anti-IGF2BP1/IMP1 antibody [EPR18791] ab184305) at 1/1000 dilution

    Lanes 1 - 3: Western blot - Anti-IMP3 antibody [EPR12021-114] (Anti-IMP3 antibody [EPR12021-114] ab179807) at 1/1000 dilution

    Lane 1: C-Myc/DDK-tagged full length Human IGF2BP1 recombinant at 0.01 µg

    Lane 2: C-Myc/DDK-tagged full length Human IGF2BP2 recombinant protein at 0.01 µg

    Lane 3: N-His-tagged full length Human IGF2BP3 recombinant protein at 0.01 µg

    Secondary

    All lanes: Western blot - Goat Anti-Rabbit IgG H&L (HRP) (Goat Anti-Rabbit IgG H&L (HRP) ab97051) at 1/20000 dilution

    Predicted band size: 63 kDa

    Observed band size: 75 kDa

    Exposure time: 180s

  • Dot Blot - Anti-DDDDK tag (Binds to FLAG® tag sequence) antibody [EPR20018-251] (ab205606), expandable thumbnail

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

    Dot blot analysis of pan NOS using Anti-pan NOS antibody [RM1126] ab316334 at 1:1000 (0.503 ug/ml) followed by a Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (Goat Anti-Rabbit IgG H&L (HRP) ab97051) at 1:20,000 dilution.

    Anti-DDDDK tag (Binds to FLAG® tag sequence) antibody [EPR20018-251] (ab205606) as total protein control staining at 1/1000 dilution.

    All lanes: Dot Blot - Anti-pan NOS antibody [RM1126] (Anti-pan NOS antibody [RM1126] ab316334) at 1/1000 dilution

    Lane 1: C-Myc/DDK tagged full length human nNOS recombinant protein

    Lane 2: C-Myc/DDK tagged full length human iNOS recombinant protein

    Lane 3: C-Myc/DDK tagged full length human eNOS recombinant protein

    Secondary

    All lanes: Dot Blot - Goat Anti-Rabbit IgG H&L (HRP) (Goat Anti-Rabbit IgG H&L (HRP) ab97051) at 1/20000 dilution

    Exposure time: 1s

  • Western blot - Anti-DDDDK tag (Binds to FLAG® tag sequence) antibody [EPR20018-251] (ab205606), expandable thumbnail

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

    Blocking and dilution buffer:5% NFDM/TBST.

    Anti-DDDDK tag (Binds to FLAG® tag sequence) antibody (ab205606) used as loading control at 1/1000 dilution. The secondary antibody used is Goat Anti-Rabbit IgG H&L (HRP) ab97051, a HRP goat anti-rabbit IgG (H+L), at a dilution of 1/20000.

    All lanes: Western blot - Anti-eIF4A3 antibody [EPR14301(B)] (Anti-eIF4A3 antibody [EPR14301(B)] ab180573) at 1/1000 dilution

    Lane 1: eIF4A1 Human Recombinant Protein at 10 ng

    Lane 2: eIF4A2 Human Recombinant Protein at 10 ng

    Lane 3: eIF4A3 Human Recombinant Protein at 10 ng

    Secondary

    All lanes: Western blot - Goat Anti-Rabbit IgG H&L (HRP) (Goat Anti-Rabbit IgG H&L (HRP) ab97051) at 1/20000 dilution

    Predicted band size: 47 kDa

    Observed band size: 46 kDa

    Exposure time: 5s

  • Western blot - Anti-DDDDK tag (Binds to FLAG® tag sequence) antibody [EPR20018-251] (ab205606), expandable thumbnail

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

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

    ab205606 was used as a loading control.

    All lanes: Western blot - Anti-iNOS+nNOS antibody [EP16634] (Anti-iNOS+nNOS antibody [EP16634] ab202417) at 1/1000 dilution

    Lane 1: C-Myc/DDK tagged full length human NOS1 (nNOS) recombinant protein at 10 ng

    Lane 2: C-Myc/DDK tagged full length human NOS2 (iNOS) recombinant protein at 10 ng

    Lane 3: C-Myc/DDK tagged full length human NOS3 (eNOS) recombinant protein at 10 ng

    Secondary

    All lanes: Western blot - Goat Anti-Rabbit IgG H&L (HRP) (Goat Anti-Rabbit IgG H&L (HRP) ab97051) at 1/20000 dilution

    Observed band size: 130 kDa, 160 kDa

    Exposure time: 20s

  • Western blot - Anti-DDDDK tag (Binds to FLAG® tag sequence) antibody [EPR20018-251] (ab205606), expandable thumbnail

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

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

    ab205606 was also used at 1/1000 dilution to detect C-Myc/DDK tagged human S6K1 and S6K2 recombinant proteins shown on the right panel.

    Anti-Caspase-9 antibody [E23] ab32539 showed cross reactivity with S6K2 in recombinant protein testing. S6K1 and S6K2 have different observed band size in endogenous and recombinant protein tests.

    Lanes 1 - 2: Western blot - Anti-S6K1 antibody [E175] (Anti-S6K1 antibody [E175] ab32359) at 1/1000 dilution

    Lanes 1 - 2: Western blot - Anti-DDDDK tag (Binds to FLAG® tag sequence) antibody [EPR20018-251] (ab205606) at 1/1000 dilution

    Lane 1: C-Myc/DDK tagged human S6K1 recombinant protein at 10 ng

    Lane 2: C-Myc/DDK tagged human S6K2 recombinant protein at 10 ng

    Secondary

    All lanes: Western blot - Goat Anti-Rabbit IgG H&L (HRP) (Goat Anti-Rabbit IgG H&L (HRP) ab97051) at 1/20000 dilution

    Predicted band size: 59 kDa

    Observed band size: 75 kDa, 60 kDa

    Exposure time: 10s

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