概述

  • 产品名称
    Anti-DDDDK tag抗体
    参阅全部 DDDDK tag 一抗
  • 描述
    兔多克隆抗体to DDDDK tag
  • 宿主
    Rabbit
  • 经测试应用
    适用于: WB, ICC/IF, ELISA, IPmore details
  • 免疫原

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

  • 常规说明
    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.

性能

  • 形式
    Liquid
  • 存放说明
    Shipped at 4°C. Store at +4°C short term (1-2 weeks). Store at -20°C or -80°C. Avoid freeze / thaw cycle.
  • 存储溶液
    Preservative: 0.1% Sodium azide
  • Concentration information loading...
  • 纯度
    Immunogen affinity purified
  • 克隆
    多克隆
  • 同种型
    IgG
  • 研究领域

应用

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.

应用 Ab评论 说明
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

靶标

  • 相关性
    This is a useful tool for the localisation and characterisation of DDDDK tagged proteins.
  • 别名
    • 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

图片

  • 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).

文献

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 52 Publications for this product

客户评价及客户问答

1-10 of 21 Abreviews or Q&A

Application
Western blot
Sample
Mouse Cell lysate - other (neuroblastoma2 a cells)
Gel Running Conditions
Reduced Denaturing (10)
Loading amount
10 µg
Specification
neuroblastoma2 a cells
Blocking step
Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: RT°C

Abcam user community

Verified customer

提交于 Mar 29 2017

Abcam has not validated the combination of species/application used in this Abreview.
Application
Immunohistochemistry (Frozen sections)
Sample
Mouse Tissue sections (brain)
Permeabilization
Yes - Triton x-100
Specification
brain
Blocking step
Serum as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 2.5% · Temperature: 25°C
Fixative
Paraformaldehyde

久仁子 島崎

Verified customer

提交于 Sep 05 2016

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

Abcam user community

Verified customer

提交于 Apr 22 2016

Application
Immunohistochemistry (PFA perfusion fixed frozen sections)
Blocking step
Serum as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 3% · Temperature: 20°C
Antigen retrieval step
None
Sample
Mouse Tissue sections (Brain- striatal medium spiny neurons)
Specification
Brain- striatal medium spiny neurons
Permeabilization
Yes - 0.03% Triton
Fixative
10% Formalin

Rivka Schwarcz

Verified customer

提交于 Jul 28 2014

Application
Immunocytochemistry/ Immunofluorescence
Blocking step
BSA as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 22°C
Sample
Monkey Cell (COS7)
Specification
COS7
Permeabilization
Yes - Tritonx 100
Fixative
Formaldehyde

Abcam user community

Verified customer

提交于 Oct 18 2013

Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Immunocytochemistry/ Immunofluorescence
Sample
Human Cell (hTERT-RPE1)
Specification
hTERT-RPE1
Fixative
Paraformaldehyde
Permeabilization
Yes - saponin
Blocking step
BSA as blocking agent for 20 minute(s) · Concentration: 0.02% · Temperature: 18°C

Abcam user community

Verified customer

提交于 Dec 19 2012

Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Western blot
Sample
Human Cell lysate - whole cell (hTERT-RPE1)
Loading amount
50000 cells
Specification
hTERT-RPE1
Treatment
transfected with flag tag plasmid 20hr
Gel Running Conditions
Reduced Denaturing (8 % acrylamid)
Blocking step
Milk as blocking agent for 12 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 4°C

Abcam user community

Verified customer

提交于 Dec 14 2012

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

1-10 of 21 Abreviews or Q&A

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

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