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

21-21 of 21 Abreviews or Q&A

Question

ANTIBODY CODE ab1162 BATCH NUMBER 50573 ORDER NUMBER -- NOT SPECIFIED -- DESCRIPTION OF THE PROBLEM No signal or weak signal SAMPLE E.coli whole cell lysate containing DDDDK-tagged (internal tags) proteins PRIMARY ANTIBODY ab1162, AbCam anti-DDDDK rabbit IgG polyclonal 1/3000. Incubated 1h, washed 3x10min Tried Bethyl's version first, same type/conditions SECONDARY ANTIBODY Sigma goat anti-rabbit IgG-HRP conj. 1/3000, 1h incubation, 3x10min washes. DETECTION METHOD Western Lightning enhanced luminol/oxidising chemiluminescence reagents POSITIVE AND NEGATIVE CONTROLS USED +ve = enterokinase cleavage control protein included in recombinant EK kit from Novagen - contains DDDDK sequence. -ve = lysate of strain not containing expression plasmid for the DDDDK-tagged protein ANTIBODY STORAGE CONDITIONS 4 degC SAMPLE PREPARATION cell pellet resuspended and boiled 5 min in SDSPAGE loading buffer, reducing conditions, no protease inhibitors AMOUNT OF PROTEIN LOADED equivalent to 100ul of overnight culture, our protein is easily seen under the same conditions with a custom polyclonal specific for the protein of interest ELECTROPHORESIS/GEL CONDITIONS 10% SDS-PAGE non-reducing TRANSFER AND BLOCKING CONDITIONS semidry transfer, 20 min. Blocked O/N 4 degC with 5% skimmed milk in TBS-0.1% Tween + 5mMCaCl2 (previously tried 3% BSA in PBS-0.1%Tween) HOW MANY TIMES HAVE YOU TRIED THE APPLICATION? 3 HAVE YOU RUN A "NO PRIMARY" CONTROL? No DO YOU OBTAIN THE SAME RESULTS EVERY TIME? Yes WHAT STEPS HAVE YOU ALTERED? changed from original BSA/PBS-tween protocol which works well for our specific custom antibody to milk/TBS-tween with 2 then 5mM CaCl2 added to the TBS-Tween ADDITIONAL NOTES We originally got a ladder pattern on the western of non-specific bands, we were advised to use TBS in place of PBS and add CaCl2 in case Ca2+ in the system is sequestered by phophate - this removed the ladder pattern but we only got very weak signal (film down 40 min) with one of our DDDDK-tagged proteins +ve control did not come up so perhaps we should try the Invitrogen positope R900-40 as described in your datasheet? Any other recommendations for tweeking the binding would be much appreciated!

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Answer

Thank you very much for your patience. I'm sorry to hear that you are experiencing trouble with this antibody. At this point I would suggest increasing the concentration of the primary antibody and incubate with the primary for a longer period of time; the orginator of ab1162 incubated for at least 120 minutes and if needed, try incubating overnight at 4C. The originator has also suggested that perhaps you try the 12-tag cell lysate marker in your system. Also, you mentioned that you tried Bethyl's antibody first - did that work for you? If you continue to experience trouble with this antibody, please do let me know.

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21-21 of 21 Abreviews or Q&A

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