Agarose Anti-DDDDK tag (Binds to FLAG® tag sequence) 抗体 (ab1240)

Submit a review Q&A (8)References (4)

Key features and details

  • Agarose Goat polyclonal to DDDDK tag (Binds to FLAG® tag sequence)
  • Suitable for: IP
  • Reacts with: Species independent
  • Conjugation: Agarose
  • Isotype: IgG

製品の概要

  • 製品名

    Agarose Anti-DDDDK tag (Binds to FLAG® tag sequence) antibody
    DDDDK tag (Binds to FLAG® tag sequence) 一次抗体 製品一覧

  • 製品の詳細

    Agarose Goat polyclonal to DDDDK tag (Binds to FLAG® tag sequence)

  • 由来種

    Goat

  • 標識

    Agarose

  • アプリケーション

    適用あり: IPmore details

  • 種交差性

    交差種: Species independent

  • 免疫原

    Full length native protein (purified) corresponding to DDDDK tag conjugated to Keyhole Limpet Haemocyanin (KLH).
    Sequence:

    xxxDDDDK

    Run BLAST with BLAST the sequence with ExPASy Run BLAST with BLAST the sequence with NCBI

  • 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.

    Affinity purified antibodies were coupled to agarose beads using a cyanogen bromide method.

    FLAG® is a registered trade mark of Sigma Aldrich Biotechnology LP. It is used here for informational purposes only.

    Reproducibility is key to advancing scientific discovery and accelerating scientists’ next breakthrough.

    Abcam is leading the way with our range of recombinant antibodies, knockout-validated antibodies and knockout cell lines, all of which support improved reproducibility.

    We are also planning to innovate the way in which we present recommended applications and species on our product datasheets, so that only applications & species that have been tested in our own labs, our suppliers or by selected trusted collaborators are covered by our Abpromise™ guarantee.

    In preparation for this, we have started to update the applications & species that this product is Abpromise guaranteed for.

    We are also updating the applications & species that this product has been “predicted to work with,” however this information is not covered by our Abpromise guarantee.

    Applications & species from publications and Abreviews that have not been tested in our own labs or in those of our suppliers are not covered by the Abpromise guarantee.

    Please check that this product meets your needs before purchasing. If you have any questions, special requirements or concerns, please send us an inquiry and/or contact our Support team ahead of purchase. Recommended alternatives for this product can be found below, as well as customer reviews and Q&As.

製品の特性

アプリケーション

Our Abpromise guarantee covers the use of ab1240 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
IP Use at an assay dependent dilution.

ターゲット情報

  • 関連性

    This is a useful tool for the localisation and characterisation of DDDDK tagged proteins (Binds to FLAG® tag sequence).

  • 別名

    • DDDDK epitope tag antibody
    • DDDK antibody
    • ddk antibody
    • DYKDDDDK (also known as the FLAG® tag sequence) 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

参考文献 (4)

ab1240 を使用した論文を発表された方は、こちらまでお知らせください。データシートに掲載させていただきます。

ab1240 は 4 報の論文で使用されています。

  • Ji L  et al. PIDD interaction with KEAP1 as a new mutation-independent mechanism to promote NRF2 stabilization and chemoresistance in NSCLC. Sci Rep 9:12437 (2019). PubMed: 31455821
  • Hughes DJ  et al. NEDDylation is essential for Kaposi's sarcoma-associated herpesvirus latency and lytic reactivation and represents a novel anti-KSHV target. PLoS Pathog 11:e1004771 (2015). IP ; Human . PubMed: 25794275
  • Klement K  et al. Opposing ISWI- and CHD-class chromatin remodeling activities orchestrate heterochromatic DNA repair. J Cell Biol 207:717-33 (2014). PubMed: 25533843
  • Ursini-Siegel J  et al. The ShcA SH2 domain engages a 14-3-3/PI3'K signaling complex and promotes breast cancer cell survival. Oncogene 31:5038-44 (2012). IP . PubMed: 22286768

レビューと Q&A

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1-8 of 8 Abreviews or Q&A

Question

Hi,

I am about to use these beads after pull down of the flag protein.

https://www.abcam.com/DDDDK-tag-antibody-Agarose-ab1240.html

I am washing them in RIPA and PBS and was wondering at which speed you recommend to centrifuge to pellet the beads. Is it fine using 14,000 g for 4 mins, or is it enough 30’’? I have just realised that as I am doing co-ip, 4 mins centrifugation may be too harsh on the protein complexes? I have used in the passed magnetic beads so I didn’t need to centrifuge.

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Answer

Thank you for contacting us.

If you are doing co-IP, it is not recommended to use RIPA buffer as it denaturing and may be too harsh on your protein complexes. I would suggest using a non-denaturing lysis buffer. For example:

20 mM Tris HCl pH 8
137 mM NaCl
1% Nonidet P-40 (NP-40) (Triton X-100 can be substituted for NP-40)
2 mM EDTA

Store up to 6 months at 4°C. Immediately before use add protease inhibitors.

As for your question regarding centrifugation, as shown on https://www.abcam.com/index.html?pageconfig=resource&rid=11385#d, section 4.2 "Method B", step 5, we suggest centrifuging at 1,000-3,000 g for 2 minutes at 4°C.

I hope this information is helpful to you. Please do not hesitate to contact us if you need any more advice or information.

Use our products? Submit an Abreview. Earn rewards!
https://www.abcam.com/abreviews

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Question

I have now received the beads, and was wondering how much to add of the 50% slurry per immunoprecipitation.

I have read on the label of the beads 100ug (therefore I assume the concentration of the antibody it will be 100ug/200ul gel which is 0.5ug/ul. If I use 20ul of beads gel +20 ul PBS per IP (50%slurry), then I will be using 10ug of antibody per IP (the protein comes from one 6-well plate for each IP). Isn’t this too much? The general protocol of Abcam says to add 70 to 100 ul of the slurry but that would be even more antibody.

I used to use 2ug of IP antibody plus 100ug of protein for IP and worked always fine, so I am wondering if 10ug won’t be too much for one IP and will result in non specific binding?? I guess it is a bit different, because here I am pulling down directly with the anti-“flag” antibody, but would like to know how much antibody I should add to the IP.

Thank you so much again for your help,

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Answer

Thank you for your reply.

I would recommend for the first time use of using a couple of different antibody concentrations and then seeing which one gives the best signal. You could have one condition where you use 2ug of antibody and another where you use 5ug of antibody.

If there is anything else I can help you with, please let me know.

Also, since you are based in the UK and I am based on the West coast of the USA, if you wish to receive faster replies, you could email mailto:technical@abcam.com and that will go to our UK team. I am more than happy to give any assistance, I just wanted to let you know why there is a delay in my response.

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Question

Thank you very much for your prompt reply. So, if I understand correctly, these are pre-swollen beads ready to use?

If I want to use a 50% slurry, does this mean that I should take 50ul of the beads (as they come in the tube), wash them a couple of times with PBS and then add 100 ul of PBS?

I have always worked with magnetic beads so I am getting a bit confused!

Thanks a lot again,

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Answer

Thank you for your reply.

This product are ready to use and the total volume of each vial you receive should be 400ul (200ul of gel and 200ul of buffer). Therefore to get a 50% slurry, you would have to use 100ul of product, wash in PBS and then resuspend in 50ul of PBS, that would give you 50ul of gel and 50ul of PBS.

If there is anything else I can help you with, please let me know.

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Question

I am going to use this product Anti-DDDDK tag antibody (Agarose) (ab1240) and I was wondering if you have any guidelines to use these beads, especially at which speed can I centrifuge them and for how long? How many times to wash the beads and which buffer to use for this purpose? Thanks a lot!

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Answer

Thank you for contacting Abcam.

You would use the product, ab1240 as you would use regular Protein A/G Sepharose beads, same type of buffers and centrifugation steps. I have attached the Abcam protocols booklet, which contains a general IP protocol that you would be able to use for guidance.

If there is anything else I can help you with, please let me know.

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Question

What's the conc? Whole antibody, the whole constuct?

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Answer

Vielen Dank für Ihren Anruf.

Da es für Sie so dringend ist, habe ich im Labor angerufen, und man konnte mir tatsächlich prompt Auskunft geben:

Die Konzentration beträgt 10 ug Antikoerper/200 ug "Slurry", das Volumen beträgt 400 ul.

Verwenden Sie 15 bis 25 mcl pro 0.1-1.0 mg Proteinlysate.

Ich hoffe, dies hilft Ihnen weiter.







Ich hoffe, dies hilft Ihnen weiter. Bitte zögern Sie nicht, sich wieder bei uns zu melden, falls Sie weitere Fragen haben.

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Question

I'd like to do Immunoprecipitation by using ab1233 and ab1240. Is that including agarose bead and antibodies?

Then, when I use that product, how could I do "pre-clear step"?
It is my first time to do IP, and as I known "pre-clear"step is needed for reducing non-specific pull down.
Please let me know if it is not necessary.

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Answer

Thank you for your inquiry.


Pre-clearing the lysates is always up to the end-user to determine because background levels will vary. Since both of these antibodies are conjugated to agarose, if you want to pre-clear the lysates you could use an isotype control.


For your information, we do have a suitable isotype control for both ab1233 and ab1240 as a goat IgG Agarose conjugated antibody, ab104155.


https://www.abcam.com/Goat-IgG-Agarose-ab104155.html


Also, here is our general IP protocol:


https://www.abcam.com/index.html?pageconfig=resource&rid=11385#c


I hope this information helps. Please contact us with any other questions.

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Question

Dear Sir/Madam,   I ordered two vials of anti  DDDDK antibody (agarose) from your company, we found that the amout of agarose beads in the first one is only half of the the second one, but they have the same lot number. So I think the first one has something wrong!!    Looking forward to hearing from you soon.

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Answer

Thank you for contacting us. I am sorry to hear you have been experiencing problems with this anti-DDDDK tag antibody. Indeed, 100 µg of the affinity purified antibody is attached to the agarose gel and the total volume of each vial you received should be 400 microliters (200 microliters of gel and 200 microliters of buffer). I am sorry that you did nor receive this for one of the vials and for the inconvenience this has caused. Therefore, I have issued a free of charge replacement with the order number 1003302 which you should receive after the christmas holidays. To check the status of the order please contact our Customer Service team and reference this number. Please note that this free of charge replacement vial is also covered by our Abpromise guarantee. Should you still be experiencing difficulties, or if you have any further questions, please do not hesitate to let us know. I wish you the best of luck with your research.

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Question

What is concentration of antibody?

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Answer

Thank you for your inquiry. Concentration: 100 ug antibody/200 ul gel Volume: 400 ul of 50% slurry containing 200 ul gel Antibody concentration was determined by extinction coefficient: absorbance at 280 nm of 1.4 equals 1.0 mg of IgG, before coupling to agarose beads. I hope this information helps. Please contact us with any other questions.

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