Anti-DDDDK tag (Binds to FLAG® tag sequence) 抗体 (ab1257)

Reviews (8)Q&A (2)References (31)

製品の概要

  • 製品名

    Anti-DDDDK tag (Binds to FLAG® tag sequence) antibody
    DDDDK tag (Binds to FLAG® tag sequence) 一次抗体 製品一覧

  • 製品の詳細

    Goat polyclonal to DDDDK tag (Binds to FLAG® tag sequence)

  • 由来種

    Goat

  • アプリケーション

    適用あり: ELISA, WB, ICC/IFmore details

  • 種交差性

    交差種: Species independent

  • 免疫原

    Synthetic peptide corresponding to DDDDK tag conjugated to Keyhole Limpet Haemocyanin (KLH).
    Sequence:

    xxxDDDDK

    Run BLAST with BLAST the sequence with ExPASy Run BLAST with BLAST the sequence with NCBI

  • 特記事項

    Epitope tags provide a method to localize gene products in a variety of cell types, study the topology of proteins and protein complexes, identify associated proteins, and characterize newly identified, low abundance or poorly immunogenic proteins when protein specific antibodies are not available. Tagging with xxxDDDDK may be done at the N-terminus, N-terminus preceded by a methionine residue, C-terminus, and in internal positions of the target protein. The small size of the epitope tag and its high hydrophilicity tend to decrease the possibility of interference with protein expression, proteolytic maturation, antigenicity and function. The enterokinase cleavage site allows it to be completely removed from the purified fusion proteins.

    FLAG® is a registered trade mark of Sigma Aldrich Biotechnology LP.

製品の特性

  • 製品の状態

    Liquid

  • 保存方法

    Shipped at 4°C. Upon delivery aliquot and store at -20°C. Avoid freeze / thaw cycles.

  • バッファー

    Preservative: 0.1% Sodium azide
  • Concentration information loading...

  • 精製度

    Immunogen affinity purified

  • ポリ/モノ

    ポリクローナル

  • アイソタイプ

    IgG

  • 研究分野

アプリケーション

Our Abpromise guarantee covers the use of ab1257 in the following tested applications.

The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.

アプリケーション Abreviews 特記事項
ELISA 1/100 - 1/500.
WB 1/1000 - 1/30000.
ICC/IF 1/200 - 1/2000.

ターゲット情報

  • 関連性

    This is a useful tool for the localisation and characterisation of DDDDK tagged proteins.

  • 別名

    • DDDDK epitope tag antibody
    • DDDK antibody
    • ddk antibody
    • DYKDDDDK antibody
    • DYKDDDDK epitope tag antibody
    • DYKDDDDK tag antibody
    • ECS epitope tag antibody
    • ECS tag antibody
    • Enterokinase Cleavage Site epitope tag antibody
    • Enterokinase Cleavage Site tag antibody
    • FLAG® antibody
    • FLAG® tag antibody
    see all

画像

  • Immunocytochemistry/ Immunofluorescence - Anti-DDDDK tag (Binds to FLAG® tag sequence) antibody (ab1257)
    Immunocytochemistry/ Immunofluorescence - Anti-DDDDK tag (Binds to FLAG® tag sequence) antibody (ab1257)

    Human microvascular endothelial cells expressing DDDDK-tagged beta-catenin following transient transfection.
    Ab1257 used at 1 µg/ml. Detected with FITC labeled rabbit anti-goat IgG (H&L) secondary antibody.

  • Western blot - Anti-DDDDK tag (Binds to FLAG® tag sequence) antibody (ab1257)
    Western blot - Anti-DDDDK tag (Binds to FLAG® tag sequence) antibody (ab1257)
    All lanes : Anti-DDDDK tag (Binds to FLAG® tag sequence) antibody (ab1257) at 0.04 µg/ml

    Lane 1 : E. coli whole cell lysate. at 0.2 µg
    Lane 2 : E. coli whole cell lysate. at 0.1 µg
    Lane 3 : E. coli whole cell lysate. at 0.05 µg


    Western Blot analysis of E. coli Whole Cell Lysates, labelled with DDDDK-tagged Protein by ab1257 at 1/25,000.

参考文献

This product has been referenced in:

See all 31 Publications for this product

ab1257 を使用した論文を発表された方は、こちらまでお知らせください。データシートに掲載させていただきます。

レビューと Q&A

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1-10 of 10 Abreviews or Q&A

Western blot abreview for Anti-DDDDK tag (Binds to FLAG® tag sequence) antibody

 Excellent
Abreviews
abreview image
Application
Western blot
Sample
Human Cell lysate - nuclear (Vascular smooth muscle cells)
Gel Running Conditions
Reduced Denaturing
Loading amount
20 µg
Specification
Vascular smooth muscle cells
Blocking step
Milk as blocking agent for 3 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 21°C
Read More

Abcam user community

Verified customer

投稿 Mar 22 2018

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) abreview for Anti-DDDDK tag (Binds to FLAG® tag sequence) antibody

 Average
Abreviews
abreview image
Application
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)
Sample
Mouse Tissue sections (Brain)
Antigen retrieval step
Heat mediated - Buffer/Enzyme Used: citrate buffer
Permeabilization
No
Specification
Brain
Blocking step
Serum as blocking agent for 30 minute(s) · Concentration: 10% · Temperature: 25°C
Fixative
Formaldehyde
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Abcam user community

Verified customer

投稿 Mar 05 2018

Western blot abreview for Anti-DDDDK tag (Binds to FLAG® tag sequence) antibody

 Below Average
Abreviews
abreview image
Application
Western blot
Sample
Human Cell lysate - whole cell (HEK293)
Gel Running Conditions
Reduced Denaturing
Loading amount
20 µg
Specification
HEK293
Blocking step
Milk as blocking agent for 30 minute(s) · Concentration: 5% · Temperature: 25°C
Read More

Abcam user community

Verified customer

投稿 Feb 28 2018

Western blot abreview for Anti-DDDDK tag (Binds to FLAG® tag sequence) antibody

 Excellent
Abreviews
abreview image
Application
Western blot
Sample
Rat Cell lysate - other (IP from Neonatal Rat Ventricular Myocytes infected)
Gel Running Conditions
Reduced Denaturing (10% SDS PAGE)
Loading amount
1.5 µg
Specification
IP from Neonatal Rat Ventricular Myocytes infected
Blocking step
Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 25°C
Read More

Dr. Chris Tracy

Verified customer

投稿 Dec 16 2014

Immunohistochemistry (Frozen sections) abreview for Anti-DDDDK tag (Binds to FLAG® tag sequence) antibody

 Good
Abreviews
abreview image
Application
Immunohistochemistry (Frozen sections)
Sample
Mouse Tissue sections (Muscle)
Permeabilization
Yes - Triton 0.5%
Specification
Muscle
Blocking step
BSA as blocking agent for 45 minute(s) · Concentration: 5% · Temperature: 20°C
Fixative
Paraformaldehyde
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Abcam user community

Verified customer

投稿 Oct 27 2014

Immunocytochemistry/ Immunofluorescence abreview for Anti-DDDDK tag (Binds to FLAG® tag sequence) antibody

 Excellent
Abreviews
abreview image
Application
Immunocytochemistry/ Immunofluorescence
Sample
Human Cell (Vascular smooth muscle cells)
Permeabilization
Yes - NP40
Specification
Vascular smooth muscle cells
Blocking step
BSA as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 3% · Temperature: 21°C
Fixative
Formaldehyde
Read More

Abcam user community

Verified customer

投稿 Dec 16 2013

Immunocytochemistry/ Immunofluorescence abreview for Anti-DDDDK tag (Binds to FLAG® tag sequence) antibody

 Good
Abreviews
abreview image
Application
Immunocytochemistry/ Immunofluorescence
Sample
Human Cell (COS7)
Permeabilization
Yes - triton X100
Specification
COS7
Blocking step
BSA as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 22°C
Fixative
Formaldehyde
Read More

Abcam user community

Verified customer

投稿 Oct 18 2013

Western blot abreview for Anti-DDDDK tag (Binds to FLAG® tag sequence) antibody

 Good
Abreviews
abreview image
Application
Western blot
Sample
Human Cell lysate - whole cell (Kidney cancer cell)
Gel Running Conditions
Non-reduced Denaturing (10% gel)
Loading amount
20 µg
Specification
Kidney cancer cell
Blocking step
Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 21°C
Read More

Abcam user community

Verified customer

投稿 Nov 12 2010

Question

I would like to detect FLAG (DDDDK) by immunohistology and wanted to know if you have an antibody that you would recommend for paraffin fixed sections. We work on bone so frozen sections are a bit of a nightmare for us.

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Answer

Thank you for your enquiry. None of our FLAG tag antibodies have currently been tested in IHC on paraffin sections. Some have been tested on frozen sections (ab1257,ab21536) and as this work in this application this indicates that those antibodies have the ability to recognise the native form of the protein so have good chances of working in other immunohistochemical methods. They therefore have a chance of working in IHC-P, providing the fixation of the tissue does not modify the epitope dramatically and prevent recognition by the antibodies. I hope this information will help you, please do not hesitate to contact me again if you need more advice,

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Question

DESCRIPTION OF THE PROBLEM High background, multiple bands and wrong band size SAMPLE mouse stomach sonicated in RIPA buffer plus complete protease inhibitors (Roche # 1697498) on ice. The whole tissue extract was separated on 15% SDS-PAGE and thansfered to PVDF membrane. PRIMARY ANTIBODY Ab1257 goat anti-ddddk 1:1000, 1: 2000, 1: 5000, 1:10000 and 1: 15000 in PBS-T with/without 5% non-fat milk incubated for 1 hr/RT wash in PBS-T 10 minx3 time SECONDARY ANTIBODY Sigma rabbit anti-goat, HRP conjugated 1:2000, 1: 5000 in PBS-T incubated for 30 min to 1 hr/RT wash in PBS-T 10 minx3 times DETECTION METHOD ECL POSITIVE AND NEGATIVE CONTROLS USED Negative control: stomach extract without my transgene, HeLa cell extract Positive control: HeLa cell with my transgene ANTIBODY STORAGE CONDITIONS 4 C cold room in original package SAMPLE PREPARATION RIPA buffer plus complete protease inhibitors (Roche # 1697498). AMOUNT OF PROTEIN LOADED 20 ug/well ELECTROPHORESIS/GEL CONDITIONS SDS-PAGE, reducing, 15% precast gel from BioRad TRANSFER AND BLOCKING CONDITIONS Stander semi-dry transfer buffer Blocking in 5% non-fat milk HOW MANY TIMES HAVE YOU TRIED THE APPLICATION? >10 HAVE YOU RUN A "NO PRIMARY" CONTROL? Yes DO YOU OBTAIN THE SAME RESULTS EVERY TIME? Yes WHAT STEPS HAVE YOU ALTERED? primary Ab dilution, secondary Ab dilution ADDITIONAL NOTES I used Sigma M2 Ab (monoclonal from mouse) as golden standard for FLAG tag detection. Now I turned to your product because it's cheaper. In the whole series of my western, The M2 Ab gives me a sharp, solid single band without background, which means there is no degradation in my sample.

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Answer

Results such as these are commonly solved by loading less lysate (try 5 mcg/lane), diluting the primary antibody more (try 1:25,000) and diluting the secondary antibody more (try 1:20,000). The goat anti-ECS is 10 to 20 fold higher titered than the Sigma M2 antibody. You could use the Sigma M2 antibody at 1:25 and would likely get results similar to those you are seeing with the goat anti-ECS.

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