Anti-DDDDK tag antibody [F-tag-01] - (Equivalent to FLAG antibodies from Sigma) (ab18230)

Overview

  • Product name
    Anti-DDDDK tag antibody [F-tag-01] - (Equivalent to FLAG antibodies from Sigma)
    See all DDDDK tag primary antibodies
  • Description
    Mouse monoclonal [F-tag-01] to DDDDK tag - (Equivalent to FLAG antibodies from Sigma)
  • Host species
    Mouse
  • Tested applications
    Suitable for: ICC, WB, ICC/IFmore details
    Unsuitable for: IP
  • Species reactivity
    Fusion proteins from all species containing the DYKDDDDK epitope.
  • Immunogen

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

    DYKDDDDK

Properties

  • Form
    Liquid
  • Storage instructions
    Shipped at 4°C. Store at +4°C short term (1-2 weeks). Upon delivery aliquot. Store at -20°C or -80°C. Avoid freeze / thaw cycle.
  • Storage buffer
    pH: 7.40
    Preservative: 0.097% Sodium azide
    Constituent: PBS
  • Concentration information loading...
  • Purity
    Protein A purified
  • Clonality
    Monoclonal
  • Clone number
    F-tag-01
  • Isotype
    IgG1
  • Research areas

Applications

Our Abpromise guarantee covers the use of ab18230 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
ICC Use a concentration of 2 - 10 µg/ml.
WB Use a concentration of 2 µg/ml. Detects a band of approximately 55 kDa (predicted molecular weight: 55 kDa).
ICC/IF Use at an assay dependent dilution.
  • Application notes
    Is unsuitable for IP.
  • 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

    • ab18230 staining DDDDK tag in Human HeLa, cervical carcinoma cells by ICC/IF (Immunocytochemistry/immunofluorescence). Cells were fixed with paraformaldehyde, permeabilized with 0.3% tween20 and blocked with 0.5% BSA for 1 hour at 22°C. Samples were incubated with primary antibody (1/200 in PBS) for 24 hours at 4°C. Ab8517 (1/200) was used as the secondary antibody

      See Abreview

    • All lanes : Anti-DDDDK tag antibody [F-tag-01] - (Equivalent to FLAG antibodies from Sigma) (ab18230) at 2 µg/ml

      Lane 1 : Hek293 cells transfected with empty vector
      Lane 2 : HEK293 cells transfected with vector overexpressing DDDDK tag

      Secondary
      All lanes : Goat anti-mouse IgG conjugated to HRP

      Performed under reducing conditions.

      Predicted band size: 55 kDa



      Blocked for 16 hours/ 4oC in 3.0% Non-fat milk, PBS, 0.1% Tween-20

      See Abreview

    • (a) Fusion of nuclear protein stained with ab18230 (red)
      (b) Cell nuclei stained with DAPI (blue)
      (c) Merge of (a) and (b) confirming nuclear localisation of the fusion protein.
    • Immunofluorescence analysis of transfected HEK293 cells, staining DDDDK tag with ab18230 at 1/200 dilution.

    References

    This product has been referenced in:
    • Velloso FJ  et al. Expression and in vitro assessment of tumorigenicity for NOD1 and NOD2 receptors in breast cancer cell lines. BMC Res Notes 11:222 (2018). Read more (PubMed: 29615116) »
    • Eva R  et al. EFA6 regulates selective polarised transport and axon regeneration from the axon initial segment. J Cell Sci 130:3663-3675 (2017). Read more (PubMed: 28935671) »
    See all 18 Publications for this product

    Customer reviews and Q&As

    1-9 of 9 Abreviews or Q&A

    Abcam guarantees this product to work in the species/application used in this Abreview.
    Application
    Immunocytochemistry/ Immunofluorescence
    Blocking step
    BSA as blocking agent for 40 minute(s) · Concentration: 5% · Temperature: 22°C
    Sample
    Monkey Cell (COS-7)
    Specification
    COS-7
    Permeabilization
    Yes - 0.2% triton
    Fixative
    Formaldehyde

    Abcam user community

    Verified customer

    Submitted Sep 12 2013

    Answer

    Merci de nous avoir contactés.

    Nous avons plusieurs anti-FLAG anticorps au catalogue par contre aucun n’a été testé en microscopie électronique.

    Cependant, je peux vous proposer :

    ab1162 (www.abcam.com/ab1162)
    ab 18230 (www.abcam.com/ab18230)
    ab21536 (www.abcam.com/ab21536)

    Ces anticorps n’ont pas encore été testés en microscopie électronique et nous n’offrons pas d’échantillon gratuit à des fins de tests préliminaires. Cependant, si vous souhaitez tester l’un de ces anticorps en microscopie électronique, il me sera possible de vous offrir une remise. Après soumission d’une Abreview avec vos résultats, un avoir de la valeur de l'anticorps testé vous sera offert.

    Étapes à suivre :

    1. Nous informer que vous souhaitez tester un de nos anticorps en microscopie électronique et nous vous enverrons un code promotionnel (valable 4 mois) à utiliser après obtention et envoi de vos résultats,

    2. Acheter l'anticorps,

    3. Nous envoyer vos résultats (positifs ou négatifs) obtenus sous forme d'Abreview (www.abcam.com/abreviews).

    4. L'envoi de vos résultats activera le code et vous donnera un avoir de la valeur de l'anticorps testé.

    J'espère que ces informations vous aideront. N'hésitez pas à me contacter si vous avez besoin de plus de renseignements.

    Read More
    Abcam guarantees this product to work in the species/application used in this Abreview.
    Application
    Western blot
    Sample
    Mouse Recombinant protein (Rear leg muscle (homogenised))
    Loading amount
    20 µg
    Specification
    Rear leg muscle (homogenised)
    Gel Running Conditions
    Reduced Denaturing (16% Tricine)
    Blocking step
    Milk as blocking agent for 16 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 22°C

    Abcam user community

    Verified customer

    Submitted Mar 04 2011

    Abcam guarantees this product to work in the species/application used in this Abreview.
    Application
    Western blot
    Sample
    Human Cell lysate - whole cell (293 cells transfected with empty vector or FH-FLAG)
    Loading amount
    20 µg
    Specification
    293 cells transfected with empty vector or FH-FLAG
    Gel Running Conditions
    Reduced Denaturing (Pierce precise protein gel 4-20%)
    Blocking step
    Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 21°C

    Mr. Antibody Solutions

    Verified customer

    Submitted Dec 17 2010

    Abcam guarantees this product to work in the species/application used in this Abreview.
    Application
    Western blot
    Sample
    Escherichia coli Cell lysate - whole cell (chimeric protein)
    Loading amount
    2 µg
    Specification
    chimeric protein
    Gel Running Conditions
    Reduced Denaturing (4-12% bis tris)
    Blocking step
    Milk as blocking agent for 16 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 22°C

    Abcam user community

    Verified customer

    Submitted Nov 17 2010

    Abcam guarantees this product to work in the species/application used in this Abreview.
    Application
    Immunocytochemistry/ Immunofluorescence
    Sample
    Human Cell (HeLa, cervical carcinoma)
    Specification
    HeLa, cervical carcinoma
    Fixative
    Paraformaldehyde
    Permeabilization
    Yes - 0,3% Tween20
    Blocking step
    BSA as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 1% · Temperature: 22°C

    Abcam user community

    Verified customer

    Submitted Mar 25 2009

    Question

    BATCH NUMBER 335513 ORDER NUMBER 7241014 DESCRIPTION OF THE PROBLEM No signal or weak signal SAMPLE 3 different samples: 1. c-terminal FLAG-BAP (15 ng), 2. wildtype Arabidopsis seedling protein extract, 3. transgenic FLAG-fusion Arabidopsis seedling protein extract PRIMARY ANTIBODY ab18230 abcam - 1:1000, 2.5 hours, wash 3X TBST 5 min DETECTION METHOD ECL POSITIVE AND NEGATIVE CONTROLS USED see above...sigma c-terminus FLAG-BAP (15ng) ANTIBODY STORAGE CONDITIONS +4 degree (just got it last week, have not frozen it yet) SAMPLE PREPARATION all in sample buffer, heat for 3 minutes, spin, load 10 microliters AMOUNT OF PROTEIN LOADED 15 ng of control protein, roughly 3 mg total plant protein for plant samples ELECTROPHORESIS/GEL CONDITIONS 13% SDS-PAGE gel TRANSFER AND BLOCKING CONDITIONS semi-dry transfer to millipore immobilon-P PVDF membrane, blocked with 5% milk in TBST for 30 minutes SECONDARY ANTIBODY GE Healthcare ECL anti-mouse IgG HRP linked whole anitbody (from sheep) HOW MANY TIMES HAVE YOU TRIED THE APPLICATION? 1 HAVE YOU RUN A "NO PRIMARY" CONTROL? No DO YOU OBTAIN THE SAME RESULTS EVERY TIME? Yes ADDITIONAL NOTES We do a lot of western blotting, and know that all of our standard reagents and procedures are working (ie secondary anitbody, ECL detection, etc). The only step we could see changing if we try this antibody again would be using more antibody (ie 1:100) or incubating longer (ie overnight), but both of these ideas seem extreme just to get the antibody to react with the positive control! My biggest question is whether we should try a polyclonal since we have had such poor success with monoclonals (two failed monoclonals from sigma...one similar to this, where the positive control didn't work, and the other had very very high non-specific background)...if it turns out that abcam agrees that this monoclonal antibody is not working properly for what we need, would it be possible to try a polyclonal instead of getting a refund?

    Read More
    Answer

    Thank you for your enquiry. I am sorry to hear that you are experiencing difficulties with this product ab18230 in western blot. Often it is possible to make suggestions that help resolve problems. We will happily offer technical support and in the event that a product is not functioning in the applications cited on the product data sheet (and the problem has been reported within 120 days of purchase), and if it appears that the antibody is at fault, a replacement/credit note/refund will be offered. I have looked through the protocols you used and have a few questions and suggestions that might help you resolve the problem. If the suggestions do not prove to be helpful, would you please be so kind to confirm the following items in order to help me better understand the cause of the problem. Are your samples known to have sufficient levels for detection? We would normally suggest using around 20-40ug of protein per well. Most customers who use milk as a blocking agent tend to have problems detecting bands. At Abcam, almost all of our products have been tested with 5% BSA. Sometimes, a different blocking buffer might cause cross reaction between the blocking agent and antibodies, therefore causing weak bands or no bands at all. I would recommend trying 5% BSA, if you have not already done so. Also, cross reaction between the antibodies with the diluent could cause the lack of signals. We would normally use 1% BSA to dilute the primary and secondary antibodies. You can also use PBST only. Can you confirm you have reduced and denatured the sample at 95oC for 10 minutes in buffer containing SDS and mercaptoethanol? This will ensure the protein is in the correct conformation to run at the correct molecular weight and be detected by the antibody. Could the no signal you are experiencing be due to poor transfer of protein to membrane? Did you check the transfer efficiency? This can be simply done with a reversible stain such as Ponceau S. We have an abreview stating good results when incubated at 4oC and blocking with BSA. I would strongly suggest trying the same conditions for better results. I am sorry for the string of questions but I hope the above recommendations may already help you. If you have already tried the above suggestions and still experience problems, please do not hesitate to contact me with details of your shipping address/purchasing agent information and I will be more than happy to offer you a free replacement antibody of your choice.

    Read More
    Abcam guarantees this product to work in the species/application used in this Abreview.
    Application
    Western blot
    Sample
    Human Cell lysate - whole cell (HEK293 transfected with a 27 kDa construct)
    Loading amount
    25 µg
    Specification
    HEK293 transfected with a 27 kDa construct
    Blocking step
    BSA as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 3%

    Abcam user community

    Verified customer

    Submitted Apr 18 2007

    Answer

    Thank you for your enquiry, we do not know if it will recognise the FLAG tag, we know this antibody will recognise recombinant proteins containing the DYKDDDK sequence.

    Read More

    Please note: All products are "FOR RESEARCH USE ONLY AND ARE NOT INTENDED FOR DIAGNOSTIC OR THERAPEUTIC USE"

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