Overview

  • Product name
    Anti-DNA PKcs (phospho T2609) antibody [10B1]
    See all DNA PKcs primary antibodies
  • Description
    Mouse monoclonal [10B1] to DNA PKcs (phospho T2609)
  • Host species
    Mouse
  • Tested applications
    Suitable for: ICC/IF, WBmore details
  • Species reactivity
    Reacts with: Human
  • Immunogen

    Synthetic peptide corresponding to DNA PKcs (phospho T2609).
    Database link: P78527

  • Positive control
    • MOLT4 nuclear extract treated with 2 to 10 Gy radiation.

Properties

Applications

Our Abpromise guarantee covers the use of ab18356 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/IF Use at an assay dependent concentration.
WB Use a concentration of 1 µg/ml. Detects a band of approximately >400 kDa (predicted molecular weight: 460 kDa). This antibody has been seen to cross-react with a protein having an approximate molecular weight of p53. However, it is not known exactly what this protein is.

Target

  • Function
    Serine/threonine-protein kinase that acts as a molecular sensor for DNA damage. Involved in DNA nonhomologous end joining (NHEJ) required for double-strand break (DSB) repair and V(D)J recombination. Must be bound to DNA to express its catalytic properties. Promotes processing of hairpin DNA structures in V(D)J recombination by activation of the hairpin endonuclease artemis (DCLRE1C). The assembly of the DNA-PK complex at DNA ends is also required for the NHEJ ligation step. Required to protect and align broken ends of DNA. May also act as a scaffold protein to aid the localization of DNA repair proteins to the site of damage. Found at the ends of chromosomes, suggesting a further role in the maintenance of telomeric stability and the prevention of chromosomal end fusion. Also involved in modulation of transcription. Recognizes the substrate consensus sequence [ST]-Q. Phosphorylates 'Ser-139' of histone variant H2AX/H2AFX, thereby regulating DNA damage response mechanism. Phosphorylates DCLRE1C, c-Abl/ABL1, histone H1, HSPCA, c-jun/JUN, p53/TP53, PARP1, POU2F1, DHX9, SRF, XRCC1, XRCC1, XRCC4, XRCC5, XRCC6, WRN, MYC and RFA2. Can phosphorylate C1D not only in the presence of linear DNA but also in the presence of supercoiled DNA. Ability to phosphorylate p53/TP53 in the presence of supercoiled DNA is dependent on C1D.
  • Sequence similarities
    Belongs to the PI3/PI4-kinase family.
    Contains 1 FAT domain.
    Contains 1 FATC domain.
    Contains 2 HEAT repeats.
    Contains 1 PI3K/PI4K domain.
    Contains 3 TPR repeats.
  • Post-translational
    modifications
    Phosphorylated upon DNA damage, probably by ATM or ATR. Autophosphorylated on Thr-2609, Thr-2638 and Thr-2647. Thr-2609 is a DNA damage-inducible phosphorylation site (inducible with ionizing radiation, IR). Autophosphorylation induces a conformational change that leads to remodeling of the DNA-PK complex, requisite for efficient end processing and DNA repair.
    S-nitrosylated by GAPDH.
  • Cellular localization
    Nucleus.
  • Information by UniProt
  • Database links
  • Alternative names
    • DNA dependent protein kinase catalytic subunit antibody
    • DNA PK catalytic subunit antibody
    • DNA-dependent protein kinase catalytic subunit antibody
    • DNA-PK catalytic subunit antibody
    • DNA-PKcs antibody
    • DNAPK antibody
    • DNAPK catalytic subunit antibody
    • DNPK 1 antibody
    • DNPK1 antibody
    • Hyper radiosensitivity of murine scid mutation, complementing 1 antibody
    • Hyperradiosensitivity complementing 1, mouse, homolog of 1 antibody
    • HYRC 1 antibody
    • HYRC antibody
    • HYRC1 antibody
    • IMD26 antibody
    • p350 antibody
    • p460 antibody
    • PKRDC antibody
    • PRKDC antibody
    • PRKDC_HUMAN antibody
    • Protein Kinase DNA Activated Catalytic Polypeptide antibody
    • XRCC 7 antibody
    • XRCC7 antibody
    see all

Images

  • MOLT4 nuclear extract was resolved by electrophoresis, transferred to nitrocellulose, and probed with anti-DNAPKcs (Thr2609) antibody. Proteins were visualized using a goat anti-mouse secondary conjugated to HRP and a chemiluminescence detection system. Lane 1, untreated MOLT4 cells. Lane 2, MOLT4 cells exposed to 2 Gy radiation. Lane 3, MOLT4 cells exposed to 10 Gy radiation. In both cases, cells were harvested 30 minutes after radiation.

    MOLT4 nuclear extract was resolved by electrophoresis, transferred to nitrocellulose, and probed with anti-DNAPKcs (Thr2609) antibody. Proteins were visualized using a goat anti-mouse secondary conjugated to HRP and a chemiluminescence detection system. Lane 1, untreated MOLT4 cells. Lane 2, MOLT4 cells exposed to 2 Gy radiation. Lane 3, MOLT4 cells exposed to 10 Gy radiation. In both cases, cells were harvested 30 minutes after radiation.
  • Untreated HeLa cells and 3 minute UV treated HeLa cells (recovered 30 minute) were fixed with 4% paraformaldehyde (PFA) and then were permeabilized with 0.5% Triton X‐100 followed by blocking with 5% FBS. The cells were then intracellularly stained with 5 μg/ml ab18356 antibody in blocking buffer overnight at 4°C followed by staining with Alexa Fluor® 488 conjugated goat anti‐mouse IgG antibody for one hour at room temperature. Nuclei were counterstained with DAPI, and F‐actin was counterstained with Phalloidin‐TRITC. The images were captured with a 40X objective.

References

This product has been referenced in:
  • Liu X  et al. Genistein sensitizes glioblastoma cells to carbon ions via inhibiting DNA-PKcs phosphorylation and subsequently repressing NHEJ and delaying HR repair pathways. Radiother Oncol N/A:N/A (2018). Read more (PubMed: 29685705) »
  • Igarashi K  et al. An Approach to Elucidate NBS1 Function in DNA Repair Using Frequent Nonsynonymous Polymorphism in Wild Medaka (Oryzias latipes) Populations. PLoS One 12:e0170006 (2017). Read more (PubMed: 28107384) »
See all 30 Publications for this product

Customer reviews and Q&As

1-10 of 23 Abreviews or Q&A

Application
Western blot
Loading amount
80 µg
Gel Running Conditions
Reduced Denaturing (8 %)
Sample
African Green Monkey Cell lysate - whole cell (CV-1 kidney epithel cells)
Specification
CV-1 kidney epithel cells
Treatment
2 Gy 10 min
Blocking step
Superblock (Thermo Scientific) as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 100% · Temperature: 21°C

Abcam user community

Verified customer

Submitted Dec 04 2013

Answer

Thank you for confirming these details and for your cooperation.
As requested, I have issued a free of charge replacement of 2 units of ab18356 with ab97611 and ab18192.
The order number ***** and the vials will send to our distributor.
To check the status of the order please contact our distributor and reference this order 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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Answer

Thank you for those precisions.
I cannot find any order corresponding to the PO numbers you sent. Did you order the antibody via a distributor? Were the orders placed recently?
Thank you for confirming this so I can send the replacement vials to our distributor who will then dispatch the antibodies to you.

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Answer

Thank you for your message and for providing this further information.
I am sorry to hear the suggestions made have not improved the results on this occasion. I appreciate the time you have spent on these experiments and would be pleased to arrange a replacement with ab4194 (www.abcam.com/ab4194) or ab97611 (www.abcam.com/ab97611).
Could you please send me the order number and the address of your lab so I can create a free of charge replacement fom the original order?
I look forward to hearing from you with details of how you would like to proceed.

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Answer

Thank you very much for your interest in ab18356, ab4194, and ab97611.

To our knowledge, ab18356, ab4194, and ab97611 have not been tested in mouse. We don't have smaller sizes of our antibodies, but I can offer a discount off a future purchase if you buy ab18356, ab4194, or ab97611 now, test it inmouse and submit feedback to us in the form of an Abreview. It doesn’t matter whether the Abreview is positive or negative, we would just really like to receive your feedback. The discount would be to the value of 1 free primary antibody.

If you are interested in this offer, please follow these steps:

1. Reply to this e-mail to let me know that you would like to proceed and test ab18356, ab4194, or ab97611in mouse, just let me know which one. I will then send a discount code. This code must be issued before purchasing ab18356, ab4194, or ab97611 so please wait for my reply before ordering.

2. Purchase ab18356, ab4194, or ab97611 either by phone, fax, or online (www.abcam.com).

3. Test it in mouse.

4. Let us know the results, positive or negative, using our Abreview system (this will take about 10 minutes and images are great if you have them!). To find out how to submit an Abreview, please visit: https://www.abcam.com/abreviews.

5. After the review is submitted to us, the discount code becomes active. Simply place your new order by phone, fax, or on the web and mention the discount code. The discount can be redeemed for anyprimary antibodyordered and the discount code is valid for 4 months after issue.

We are always pleased to obtain feedback about our products and any information is greatly appreciated! Even if ab18356, ab4194, or ab97611 turns out to be unsuitable for mouse, you will still receive the discount on your next purchase after your Abreview has been submitted.

Please let me know if you have any questions about this offer and I would be happy to help you further.

The Terms and Conditions of this offer can be found at: www.abcam.com/collaborationdiscount.

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Answer

Thank you for your phone call. Unfortunately, your order had already shipped and we were unable to change the vial for you. I am sending you a FedEx return shipping label that you can use to send the box back to us when it arrives. I am also arranging to have the correct vial shipped to you on order ***.


DISCOUNT CODE: ***

Expiration date: September 8th, 2012

I am very pleased to hear you would like to accept our offer and test ab18356 in mouse. This code will give you: 1 free primary antibody before the expiration date. To redeem this offer, please submit an Abreview for mouse and include this code in the “Additional Comments” section so we know the Abreview is for this promotion. For more information on how to submit an Abreview, please visit the site: https://www.abcam.com/Abreviews.

Remember, we publish both positive and negative Abreviews on our datasheets so please submit the results of your tests. The code will be active once the Abreview has been submitted and can be redeemed in one of the following ways: 1) Call to place your order and mention the code to our customer service department; 2) Include the code in your fax order; 3) Place your order on the web and enter the promotional code.

Any feedback that you can provide will be greatly appreciated, whether positive or negative. If you have any further questions, please do not hesitate to contact us. We look forward to receiving your Abreview and wish you luck with your research.

The terms and conditions applicable to this offer can be found here: www.abcam.com/collaborationdiscount.

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Answer

Thank you for those precisions.
Yes, I think a transfer overnight at 30V followed by a "boost" at 70-80V for 1 hour at the end of the transfer can be of significant improvement. I would recommend to add 0.1% SDS in the transfer buffer.
A gradient gel 6-20% should be fine. Alternatively you can use a gel at 8%.
The sample preparation can maybe be shortened, as shown in the example on the datasheet the cells were exposed to up to 10 Gy radiation and then harvested for 30 minutes only after radiation : https://www.abcam.com/dna-pkcs-phospho-t2609-antibody-10b1-ab18356.html#ab18356_1.jpg
As explained in my previous email, the use of a blocking solution with BSA to block the membrane and to dilute the antibodies (primary and secondary) is also recommended.
I hope this helps.

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Question
Answer

Thank you for taking time to complete our questionnaire. I am sorry to hear that you are experiencing some problems with this antibody in Western Blot.
The details provided will enable us to investigate this case and will provide us with vital information for monitoring product quality.
Having reviewed this case, I would like to offer some suggestions to help optimize the results from ab18356 :
- I would recommend to use BSA (up to 5%) all along the protocol. It is not recommend to use 2 different blocking agents for the blocking and for the incubation of the antibodies.
- Furthermore, milk is not recommended for studies of phospho-proteins because it contains casein which is a phospho-protein.
- I would also suggest to add fresh protease and phosphatase inhibitors to the lysis buffer when you prepare your samples prior to electrophoresis.
Western blots of big proteins such as DNA PKcs can be difficult and I think some changes for the transfer may solve your problems:
- I would highly suggest to optimize the transfer conditions by increasing the transfer time to 18 or 24 hours (in some examples, for big proteins, the transfer is done for 48 hours)
- A transfer at 25-30V for a long period of time is fine but it has been shown that a "boost" at 70-80V for 1 hour at the end of the transfer can be of significant improvement.
- You can also use SDS in the transfer buffer, up to 0.1% and reduce the proportion of methanol to 10% or even less. The reason is that methanol washes out the SDS from the protein.
Should the suggestions not improve the results, please do let me know.
In the event that a product is not functioning in the species and applications cited on the product datasheet (and the problem has been reported within 6 months of purchase), we would be pleased to provide a free of charge replacement, credit note, or refund.
I hope this information is helpful, and I thank you for your cooperation.

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Answer

Vielen Dank für diese Rückmeldung! Wir würden uns über ein Feedback von Ihnen, wie die Versuche mit ab18356 geklappt haben, sehr freuen: In Ihrem Fall könnte ich nämlich eine Ausnahme machen und Ihnen den Rabattcode auch noch nachträglich ausstellen. Sie bekämen für Ihre Rezension nicht nur einen weiteren Antikörper geschenkt, sondern auch noch Abpoints, die Sie gegen Rabatte beim Kauf weiterer Produkte oder Geschenkgutscheine wie z.B. Amazon.com-Gutscheine eintauschen können. Ich wünsche Ihnen viel Erfolg für Ihr Projekt. Falls Sie einen Rabattcode erhalten möchten oder weitere Fragen haben, zögern Sie bitte nicht, sich wieder an mich zu wenden.  

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Question
Answer

Vielen Dank für Ihren Anruf und für Ihr Interesse an unseren Produkten. Leider konnte ich Sie in den letzten Tagen telefonisch nicht erreichen. Daher hoffe ich, mit dieser email Ihre Frage beantworten zu können: Unseres Wissens nach wurde der anti-DNA PKcs Antikörper ab18356 bisher noch nicht in der Maus getestet, aufgrund einer Sequenzhomologie des Immunogens von 88% ist eine Kreuzreaktivität jedoch sehr wahrscheinlich. Falls Sie dies selbst testen möchten, kann ich Ihnen zur Zeit ein spezielles Angebot über einen 100%igen Abreview-Rabatt anbieten. Bei diesem Angebot bekommen Sie einen Rabatt für eine zukünftige Bestellung, wenn Sie uns ein Abreview mit dem Testresultat zusenden. Der Rabatt würde gegen eine erneute Bestellung eines weiteren primären Antikörpers von uns verrechnet werden. Um von diesem Angebot profitieren zu können, folgen Sie bitte diesen Schritten: 1.) Bestätigen Sie mir bitte, dass Sie ab18356 in der Maus testen möchten. 2.) Bitte bestellen Sie erst nach Erhalt des Rabattcodes. 3.) Bestellen Sie den Antikörper wie üblich per Telefon, Email oder Fax 4.) Testen Sie den Antikörper in der Maus. 5.) Teilen Sie uns das Ergebnis Ihres Tests durch ein Abreview mit und notieren Sie den Discount Code in dem Feld "additional notes" Unter der folgenden URL können Sie mehr über unser Abreview System erfahren: https://www.abcam.com/abreviews 6.) Der Rabattcode ist nach dem Abschicken des Abreviews aktiv, und Sie können einen anderen primären Antikörper bei uns bestellen (halten Sie bei der Bestellung bitte den Rabattcode und die Bestellnummer bereit). Bitte beachten Sie, dass der Rabattcode innerhalb von 4 Monaten nach Ausstellung eingelöst werden muss. Der Rabattcode wird gültig unabhängig davon, ob Ihr Ergebnis positiv oder negativ ist. Die Bedingungen zu unserem 100% Abreview Rabatt können Sie unter dem folgenden Link nachlesen: https://www.abcam.com/collaborationdiscount Ich hoffe, diese Informationen helfen Ihnen weiter. Falls Sie einen Rabattcode erhalten möchten oder weitere Fragen haben, zögern Sie bitte nicht, sich wieder an mich zu wenden.

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1-10 of 23 Abreviews or Q&A

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