Anti-DDDDK tag antibody (ab1162)

Rabbit polyclonal DDDDK tag antibody. Validated in WB, IP, ELISA, ICC/IF. Cited in 61 publication(s). Independently reviewed in 15 review(s).

Overview

  • Product name
  • Description
    Rabbit polyclonal to DDDDK tag
  • Host species
    Rabbit
  • Tested applications
    Suitable for: WB, ICC/IF, ELISA, IPmore details
  • Immunogen

    Peptide xxxDDDDK conjugated to KLH. xxx represents random amino acids. This sequence represents the Enterokinase Cleavage Site.

  • 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. Store at +4°C short term (1-2 weeks). Store at -20°C or -80°C. Avoid freeze / thaw cycle.
  • Storage buffer
    pH: 7.2
    Preservative: 0.1% Sodium azide
    Constituent: PBS
  • Concentration information loading...
  • Purity
    Immunogen affinity purified
  • Clonality
    Polyclonal
  • Isotype
    IgG
  • Research areas

Applications

Our Abpromise guarantee covers the use of ab1162 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
WB Use a concentration of 0.2 - 1 µg/ml.

Antibody was analyzed by western blot using various amino-terminal and carboxy-terminal DDDDK fusion proteins and Invitrogen Positope" R900-40. A 1/5000 dilution gave bands against 0.1, 1.0 and 10 ng of the fusion proteins and 100 ng and 25 ng of the Positope.

ICC/IF Use at an assay dependent concentration.
ELISA Use a concentration of 1 - 5 µg/ml.

As coating antibody in sELISA.

IP 1/40. PubMed: 25419851

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

  • Immunofluorescent analysis of A293 cells labeling Flag-VPS35 with ab1162 at 10 μg/ml. The nuclear counter stain is DAPI (blue).

  • ab1162 staining DDDDK tag in hTERT-RPE1 by ICC/IF (Immunocytochemistry/immunofluorescence). Cells were fixed with Paraformaldehyde, permeabilized with Saponin and blocked with 0.02% BSA for 20 minutes at 18°C. Samples were incubated with primary antibody (1/100) for 45 minutes at 18°C. A Cy3®-conjugated Donkey anti-rabbit polyclonal was used as the secondary antibody.

    See Abreview

  • ab1162 staining DDDDK tag (pink) in neonatal rat cardiomyocytes transduced with Ad-TBX18/FLAG-IRES-GFP by Immunocytochemistry/ Immunofluorescence.

    Cells were fixed with paraformaldehyde, permeabilized with 0.1% Triton X-100 and incubated with primary antibody. An AlexaFluor®-conjugated anti-rabbit IgG was used as the secondary antibody. Nuclei were stained with DAPI (blue).

References

This product has been referenced in:
  • Khoury G  et al. HIV latency reversing agents act through Tat post translational modifications. Retrovirology 15:36 (2018). WB . Read more (PubMed: 29751762) »
  • Rogers OC  et al. PSA-selective activation of cytotoxic human serine proteases within the tumor microenvironment as a therapeutic strategy to target prostate cancer. Oncotarget 9:22436-22450 (2018). Read more (PubMed: 29854290) »
See all 61 Publications for this product

Customer reviews and Q&As

11-20 of 21 Abreviews or Q&A

Application
Western blot
Sample
Human Cell lysate - whole cell (HeLA)
Loading amount
25 µg
Specification
HeLA
Gel Running Conditions
Reduced Denaturing (10)
Blocking step
Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 20°C

Abcam user community

Verified customer

Submitted Jan 21 2012

Answer

Thank you very much for getting back to me so quickly. There seems to be some discrepancy which may cause the problem. The primary antibody (ab1162) is rabbit polyclonal to DDDDK tag and the secondary antibody is goat anti-mouse. The primary and the secondary antibodies are not compatible. Since the osteoclast cells are from mouse bone marrow, it may well be that the secondary antibody (goat anti-mouse) recognizes murine endogenous immunoglobulins causing the non-specific high background signal. If you need any further assistance in the future, please do not hesitate to contact me.          

Read More
Application
Western blot
Sample
Mouse Recombinant protein (Flag-tagged recombinant protein)
Loading amount
2 µg
Specification
Flag-tagged recombinant protein
Gel Running Conditions
Reduced Denaturing (4-20% Tris-Glycine Gel)
Blocking step
Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 4°C

Abcam user community

Verified customer

Submitted Aug 06 2010

Application
Western blot
Sample
Human Cell lysate - whole cell (Transfected HT1080 Cells)
Loading amount
40000 cells
Specification
Transfected HT1080 Cells
Gel Running Conditions
Reduced Denaturing (10% SDS-PAGE)
Blocking step
Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 22°C

Abcam user community

Verified customer

Submitted Feb 01 2010

Application
Immunocytochemistry
Sample
African Green Monkey Cultured Cells (Cos7 cells)
Specification
Cos7 cells
Fixative
Paraformaldehyde
Permeabilization
Yes - 10 min in 0.1% PBS-tween20
Blocking step
Serum as blocking agent for 30 minute(s) · Concentration: 10% · Temperature: 22°C

Abcam user community

Verified customer

Submitted Apr 16 2009

Application
Western blot
Sample
Rabbit Cell lysate - whole cell (HCT116)
Loading amount
30 µg
Specification
HCT116
Gel Running Conditions
Reduced Denaturing
Blocking step
Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 21°C

Dr. Emil Kofod-Olsen

Verified customer

Submitted Dec 21 2007

Application
Immunocytochemistry/ Immunofluorescence
Sample
Rabbit Cell (HCT116)
Specification
HCT116
Fixative
Formaldehyde
Permeabilization
Yes - 0.2% Triton-X 100
Blocking step
BSA as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 21°C

Dr. Emil Kofod-Olsen

Verified customer

Submitted Oct 09 2007

Application
Western blot
Sample
Human Cell lysate - whole cell (Mono Mac 1)
Loading amount
50 µg
Specification
Mono Mac 1
Blocking step
Milk as blocking agent for 2 hour(s) and 0 minute(s) · Concentration: 5

Mr. Craig Faulkner

Verified customer

Submitted Mar 20 2006

Application
Western blot
Sample
Human Cell lysate - whole cell (HEK 293)
Specification
HEK 293
Blocking step
Milk as blocking agent for 30 minute(s) · Concentration: 5%

Mr. Alexander Schepsky

Verified customer

Submitted Jan 05 2006

Answer

Thank you for your e-mail regarding our DDDDK tag antibodies. Neither antibodies have been tested for internalisation experiments and it is possible that the tag you have inserted in your protein is "embedded"/hidden in the 3D conformation of your membrane protein of interest, hence ab1162 does not recognise it and ab1259 may have the same problem. We do not routinely offer free or trial sized samples for testing purposes. Our policy at Abcam is that if an antibody does not work as specified on the datasheet, we will offer a replacement or reimbursement. Should you decide to test an antibody in an application for which we do not have any information, please let us know how you get on and in return we will award you 50 points with the Abcam Loyalty Scheme which can be redeemed on a number of rewards.

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11-20 of 21 Abreviews or Q&A

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