Anti-DDDDK tag 抗体 - (Equivalent to FLAG antibodies from Sigma) (ab1162)

製品の概要

  • 製品名

    Anti-DDDDK tag antibody - (Equivalent to FLAG antibodies from Sigma)
    DDDDK tag 一次抗体 製品一覧
  • 製品の詳細

    Rabbit polyclonal to DDDDK tag - (Equivalent to FLAG antibodies from Sigma)
  • 由来種

    Rabbit
  • アプリケーション

    適用あり: WB, ICC/IF, ELISA, IPmore details
  • 種交差性

    交差種: Species independent
  • 免疫原

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

  • 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.

製品の特性

  • 製品の状態

    Liquid
  • 保存方法

    Shipped at 4°C. Store at +4°C short term (1-2 weeks). Store at -20°C or -80°C. Avoid freeze / thaw cycle.
  • バッファー

    pH: 7.2
    Preservative: 0.1% Sodium azide
    Constituent: PBS
  • 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 ab1162 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
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

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

  • 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).

参考文献

This product has been referenced in:

  • Li Y  et al. Fungal acetylome comparative analysis identifies an essential role of acetylation in human fungal pathogen virulence. Commun Biol 2:154 (2019). Read more (PubMed: 31069264) »
  • Frattini V  et al. A metabolic function of FGFR3-TACC3 gene fusions in cancer. Nature 553:222-227 (2018). Read more (PubMed: 29323298) »
See all 67 Publications for this product

レビューと Q&A

1-6 of 6 Q&A

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

Answer

Gracias por tu confirmacion.


Acabo de hacer un nuevo pedido del anticuerpo ab1259 de forma totalmente gratuita. El número de pedido es XXXX. A lo largo del día recibirás una confirmación automática del mismo, indicando el plazo de entrega y los detalles. Si no lo recibierais, o si tuvierais alguna consulta sobre este pedido, por favor, no olvidéis mencionar el número indicado.

Espero que este nuevo anticuerpo funcione perfectamente en vuestra aplicación, y en caso contrario, por favor, no dudes en volvernos a contactar. Te recuerdo que todos los productos de Abcam tienen una garantía de 6 meses, pasada la cual no es posible ofrecer reemplazos o reembolsos. Por ello, si tuvierais algún tipo de problema, te animo a que lo reportes lo más rápido posible.

Si necesitáis cualquier otra información, o tenéis mas consultas o sugerencias, por favor, no dudéis en contactarnos de nuevo.

Read More

Answer

Te agradezco mucho que nos hayas facilitado mas detalles del protocolo y te hayas tomado la molestia de enviar las imágenes.

Es evidente que el anticuerpo no es especifico contra el DDDDK tag visto que da la misma señal en células transfectas y en células sin el tag unido a la proteína. Es curioso, sin embargo, que si lo sea en WB e IP. ¿Habéis utilizado el mismo control negativo en WB?

Me comentas que este problema lo tuvisteis en el pasado, en el momento de adquirir el anticuerpo, por lo que entiendo que podemos descartar que el problema se deba a la perdida de actividad debido a las condiciones de almacenamiento del mismo a lo largo del tiempo (para mantener en perfecto estado los anticuerpos durante más de un año, se recomienda guardarlos a -20C).

El protocolo parece perfecto, no creo que ninguna mejora que pueda hacerse haga obtener una señal más específica. En estos casos es importante comprobar que tanto la transfección del plásmido a la célula se ha realizado con éxito, así como la inserción del tag en la proteína diana. En vuestro caso lo habéis comprobado con otras técnicas, por lo que no cabe duda acerca de ésto.

Quizás ayudaría a mejorar el resultado intensificar las condiciones de permeabilización de las células. Solemos recomendar si se usa Saponin, una concentración al 0.5% durante unos diez minutos.





Así que quedo a la espera de que me indiques la mejor solución para vosotros. Si tienes cualquier otra consulta, por favor, no dudes en volver a contactarnos.

Read More

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

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.

Read More

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!

Read More
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.

Read More

Please note: All products are "FOR RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC PROCEDURES"
For licensing inquiries, please contact partnerships@abcam.com

登録