Anti-DDDDK tag antibody - (Equivalent to FLAG antibodies from Sigma) (ab1257)

Overview

  • Product name
    Anti-DDDDK tag antibody - (Equivalent to FLAG antibodies from Sigma)
    See all DDDDK tag primary antibodies
  • Description
    Goat polyclonal to DDDDK tag - (Equivalent to FLAG antibodies from Sigma)
  • Host species
    Goat
  • Tested applications
    Suitable for: ELISA, WB, ICC/IFmore details
  • Immunogen

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

    xxxDDDDK

  • General notes
    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.

Properties

  • Form
    Liquid
  • Storage instructions
    Shipped at 4°C. Upon delivery aliquot and store at -20°C. Avoid freeze / thaw cycles.
  • Storage buffer
    Preservative: 0.1% Sodium azide
  • Concentration information loading...
  • Purity
    Immunogen affinity purified
  • Clonality
    Polyclonal
  • Isotype
    IgG
  • Research areas

Applications

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.

Application Abreviews Notes
ELISA 1/100 - 1/500.
WB 1/1000 - 1/30000.
ICC/IF 1/200 - 1/2000.

Target

  • Relevance
    This is a useful tool for the localisation and characterisation of DDDDK tagged proteins.
  • Alternative names
    • 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

Images

  • 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.

  • All lanes : Anti-DDDDK tag antibody - (Equivalent to FLAG antibodies from Sigma) (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.

References

This product has been referenced in:
  • Martin LJ  et al. Electric fields control the orientation of peptides irreversibly immobilized on radical-functionalized surfaces. Nat Commun 9:357 (2018). Read more (PubMed: 29367659) »
  • Gupta S  et al. Herpesvirus deconjugases inhibit the IFN response by promoting TRIM25 autoubiquitination and functional inactivation of the RIG-I signalosome. PLoS Pathog 14:e1006852 (2018). Read more (PubMed: 29357390) »
See all 27 Publications for this product

Customer reviews and Q&As

1-10 of 10 Abreviews or Q&A

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

Abcam user community

Verified customer

Submitted Mar 22 2018

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

Abcam user community

Verified customer

Submitted Mar 05 2018

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

Abcam user community

Verified customer

Submitted Feb 28 2018

Application
Western blot
Loading amount
1.5 µg
Gel Running Conditions
Reduced Denaturing (10% SDS PAGE)
Sample
Rat Cell lysate - other (IP from Neonatal Rat Ventricular Myocytes infected)
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

Dr. Chris Tracy

Verified customer

Submitted Dec 16 2014

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

Abcam user community

Verified customer

Submitted Oct 27 2014

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

Abcam user community

Verified customer

Submitted Dec 16 2013

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

Abcam user community

Verified customer

Submitted Oct 18 2013

Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Western blot
Sample
Human Cell lysate - whole cell (Kidney cancer cell)
Loading amount
20 µg
Specification
Kidney cancer cell
Gel Running Conditions
Non-reduced Denaturing (10% gel)
Blocking step
Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 21°C

Abcam user community

Verified customer

Submitted Nov 12 2010

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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