Name: Anti-DNA PKcs antibody [Y393]
Brand: RabMAb
SKU: ab32566
Rating: 5 (13 reviews)

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Western blot - Anti-DNA PKcs antibody [Y393] (ab32566)

Key features and details

Produced recombinantly (animal-free) for high batch-to-batch consistency and long term security of supply
Rabbit monoclonal [Y393] to DNA PKcs
Suitable for: WB, IHC-P, ICC/IF
Knockout validated
Reacts with: Human

Product name

Anti-DNA PKcs antibody [Y393]
See all DNA PKcs primary antibodies
Description

Rabbit monoclonal [Y393] to DNA PKcs
Host species

Rabbit
Specificity

This antibody is specific for DNA PKcs. It may also detect the splice isoform 2.

Mouse and rat species are recommended based on WB results, we do not guarantee IHC-P for mouse and rat.

Tested Applications & Species

Application	Species
ICC/IF	Human
IHC-P	Human
WB	Human

See all applications and species data

Immunogen

Synthetic peptide within Human DNA PKcs aa 4050 to the C-terminus (C terminal). The exact sequence is proprietary.
Database link: P78527
Positive control
- WB: K562 cell lysate. ICC/IF: Hela cells. IHC-P: Human breast carcinoma tissue slides.
General notes
Mouse and rat samples are recommended based on WB results.
This product is a recombinant monoclonal antibody, which offers several advantages including:
- - High batch-to-batch consistency and reproducibility
- - Improved sensitivity and specificity
- - Long-term security of supply
- - Animal-free production
For more information see here.
Our RabMAb^® technology is a patented hybridoma-based technology for making rabbit monoclonal antibodies. For details on our patents, please refer to RabMAb^® patents.
We are constantly working hard to ensure we provide our customers with best in class antibodies. As a result of this work we are pleased to now offer this antibody in purified format. We are in the process of updating our datasheets. The purified format is designated 'PUR' on our product labels. If you have any questions regarding this update, please contact our Scientific Support team.
The Life Science industry has been in the grips of a reproducibility crisis for a number of years. Abcam is leading the way in addressing the problem with our range of recombinant monoclonal antibodies and knockout edited cell lines for gold-standard validation.

One factor contributing to the crisis is the use of antibodies that are not suitable. This can lead to misleading results and the use of incorrect data informing project assumptions and direction. To help address this challenge, we have introduced an application and species grid on our primary antibody datasheets to make it easy to simplify identification of the right antibody for your needs.

Learn more here.

Form

Liquid
Storage instructions

Shipped at 4°C. Upon delivery aliquot and store at -20°C. Avoid freeze / thaw cycles.
Storage buffer

pH: 7.20
Preservative: 0.01% Sodium azide
Constituents: 59% PBS, 40% Glycerol, 0.05% BSA
Concentration information loading...
Purity

Protein A purified
Clonality

Monoclonal
Clone number

Y393
Isotype

IgG
Research areas

Alternative Versions
- Anti-DNA PKcs antibody [Y393] - BSA and Azide free (ab174575)
- HRP Anti-DNA PKcs antibody [Y393] (ab195537)
Compatible Secondaries
Isotype control
- Rabbit IgG, monoclonal [EPR25A] - Isotype Control (ab172730)
Recombinant Protein
- Recombinant Human DNA PKcs protein (Tagged) (ab204152)

The Abpromise guarantee

Our Abpromise guarantee covers the use of ab32566 in the following tested applications.

The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.

Guaranteed

Tested applications are guaranteed to work and covered by our Abpromise guarantee.

Predicted

Predicted to work for this combination of applications and species but not guaranteed.

Incompatible

Does not work for this combination of applications and species.

Application	Species
ICC/IF	Human
IHC-P	Human
WB	Human
All applications	Mouse Rat Armenian hamster

Application	Abreviews	Notes
WB	(10)	1/1000 - 1/10000. Detects a band of approximately 460 kDa (predicted molecular weight: 469 kDa). For unpurified, use 1/1000 - 1/2000.
IHC-P		1/50. Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol. For unpurified, use 1/5. Mouse and rat species are recommended based on WB results, we do not guarantee IHC-P for mouse and rat.
ICC/IF	(2)	1/100. For unpurified, use 1/10.

Notes
WB 1/1000 - 1/10000. Detects a band of approximately 460 kDa (predicted molecular weight: 469 kDa). For unpurified, use 1/1000 - 1/2000.
IHC-P 1/50. Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol. For unpurified, use 1/5. Mouse and rat species are recommended based on WB results, we do not guarantee IHC-P for mouse and rat.
ICC/IF 1/100. For unpurified, use 1/10.

Application notes

Is unsuitable for Flow Cyt or IP.

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.
Target information above from: UniProt accession P78527 The UniProt Consortium
The Universal Protein Resource (UniProt) in 2010
Nucleic Acids Res. 38:D142-D148 (2010) .

Information by UniProt
Database links
- Entrez Gene: 5591 Human
- Entrez Gene: 19090 Mouse
- Entrez Gene: 360748 Rat
- Omim: 600899 Human
- SwissProt: P78527 Human
- SwissProt: P97313 Mouse
- Unigene: 491682 Human
- Unigene: 71 Mouse
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

Western blot - Anti-DNA PKcs antibody [Y393] (ab32566)

Lane 1: Wild-type HAP1 cell lysate (20 µg)
Lane 2: DNA PKcs knockout HAP1 cell lysate (20 µg)
Lane 3: K562 cell lysate (20 µg)
Lane 4: SH-SY5Y cell lysate (20 µg)
Lanes 1 - 4: Merged signal (red and green). Green - ab32566 observed at 470 kDa. Red - loading control, ab7291, observed at 52 kDa.

ab32566 was shown to specifically react with DNA PKCs in wild-type HAP1 cells. No band was observed when DNA PKcs knockout samples were examined. Wild-type and DNA PKCs knockout samples were subjected to SDS-PAGE. ab32566 and ab7291 (loading control to alpha tubulin) were diluted 1/1000 and 1/10,000 and incubated overnight at 4°C. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preadsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preadsorbed (ab216776) secondary antibodies at 1/10,000 dilution for 1 hour at room temperature before imaging.
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-DNA PKcs antibody [Y393] (ab32566)

Immunohistochemical staining of paraffin embedded human tonsil with purified ab32566 at a working dilution of 1 in 50. The secondary antibody used is a HRP polymer for rabbit IgG. The sample is counter-stained with hematoxylin. Antigen retrieval was perfomed using Tris-EDTA buffer, pH 9.0.
Immunocytochemistry/ Immunofluorescence - Anti-DNA PKcs antibody [Y393] (ab32566)

Immunofluorescent staining of HeLa cells (fixed with 4% PFA) with purified ab32566 at a dilution of 1/100. An Alexa Fluor® 555 goat anti-rabbit antibody was used as the secondary at a dilution of 1/200. The panel on the right shows the DAPI counter-staining.
Western blot - Anti-DNA PKcs antibody [Y393] (ab32566)

All lanes : Anti-DNA PKcs antibody [Y393] (ab32566) at 1/7300 dilution (purified)

Lane 1 : MOLT4 cell lysate
Lane 2 : HeLa cell lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : HRP goat anti-rabbit (H+L) at 1/1000 dilution

Predicted band size: 469 kDa
Observed band size: 460 kDa why is the actual band size different from the predicted?

Blocking buffer: 5% NFDM/TBST
Dilution buffer: 5% NFDM/TBST
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-DNA PKcs antibody [Y393] (ab32566)

Immunohistochemical staining of paraffin embedded human tonsil with unpurified ab32566 at a working dilution of 1 in 5. The secondary antibody used is a HRP polymer for rabbit IgG. The sample is counter-stained with hematoxylin. Antigen retrieval was perfomed using Tris-EDTA buffer, pH 9.0.
Immunocytochemistry/ Immunofluorescence - Anti-DNA PKcs antibody [Y393] (ab32566)

Immunofluorescent staining of HeLa cells (fixed with 4% PFA) with unpurified ab32566 at a dilution of 1/10. An Alexa Fluor^® 555 goat anti-rabbit antibody was used as the secondary at a dilution of 1/200. The panel on the right shows the DAPI counter-staining.
Western blot - Anti-DNA PKcs antibody [Y393] (ab32566)

All lanes : Anti-DNA PKcs antibody [Y393] (ab32566) at 1/1000 dilution (unpurified)

Lane 1 : MOLT4 cell lysate
Lane 2 : HeLa cell lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : HRP goat anti-rabbit (H+L) at 1/1000 dilution

Predicted band size: 469 kDa
Observed band size: 460 kDa why is the actual band size different from the predicted?

Blocking buffer: 5% NFDM/TBST
Dilution buffer: 5% NFDM/TBST
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-DNA PKcs antibody [Y393] (ab32566)

Unpurified ab32566, at a 1/50 dilution, staining DNA PKcs in paraffin embedded human breast carcinoma tissue by immunohistochemistry.
Western blot - Anti-DNA PKcs antibody [Y393] (ab32566)

Anti-DNA PKcs antibody [Y393] (ab32566) at 1/1000 dilution (unpurified) + K562 cell lysate.

Predicted band size: 469 kDa
Observed band size: 460 kDa why is the actual band size different from the predicted?
Anti-DNA PKcs antibody [Y393] (ab32566)

To our knowledge, customised protocols are not required for this product. Please try the standard protocols listed below and let us know how you get on.

Click here to view the general protocols

SDS download

Country/Region

Language
Datasheet download

Download

Publishing research using ab32566? Please let us know so that we can cite the reference in this datasheet.

ab32566 has been referenced in 15 publications.

Yang Q et al. Minichromosome maintenance 3 promotes hepatocellular carcinoma radioresistance by activating the NF-?B pathway. J Exp Clin Cancer Res 38:263 (2019). PubMed: 31208444
Sekiba K et al. DHX9 regulates production of hepatitis B virus-derived circular RNA and viral protein levels. Oncotarget 9:20953-20964 (2018). PubMed: 29765512
Xiao X et al. DNA-PK inhibition synergizes with oncolytic virus M1 by inhibiting antiviral response and potentiating DNA damage. Nat Commun 9:4342 (2018). PubMed: 30337542
Sun JS & Yang XH Expression of DNA-dependent protein kinase catalytic subunit in laryngeal squamous cell carcinoma and its importance. Exp Ther Med 15:3295-3301 (2018). PubMed: 29545847
Zhang H et al. CAF-secreted CXCL1 conferred radioresistance by regulating DNA damage response in a ROS-dependent manner in esophageal squamous cell carcinoma. Cell Death Dis 8:e2790 (2017). PubMed: 28518141

View all Publications for this product

Customer reviews and Q&As

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1-10 of 14 Abreviews or Q&A

Application

Western blot

Sample

Human Cell lysate - whole cell (HeLa)

Loading amount

20 µg

Specification

HeLa

Gel Running Conditions

Reduced Denaturing

Blocking step

Milk as blocking agent for 2 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 4°C

Abcam user community

Verified customer

Submitted Dec 30 2010

Application

Immunocytochemistry/ Immunofluorescence

Sample

Human Cell (breast cancer cell)

Specification

breast cancer cell

Fixative

Paraformaldehyde

Permeabilization

Yes - 0.5% triton X100

Blocking step

BSA as blocking agent for 30 minute(s) · Concentration: 3% · Temperature: 25°C

Abcam user community

Verified customer

Submitted May 09 2008

Application

Western blot

Sample

Human Cell lysate - nuclear (Vascular smooth muscle cells)

Gel Running Conditions

Reduced Denaturing

Loading amount

25 µg

Specification

Vascular smooth muscle cells

Blocking step

Milk as blocking agent for 3 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 21°C

Abcam user community

Verified customer

Submitted Nov 29 2017

Question

Purchasing 1549-1 lot YG021806C and 1579-1 lot YC120107R
The Epitomics datasheets says the lot numbers can be used to find the isotype control of the correct concentration.
Please confirm which isotype controls.

Abcam community

Verified customer

Asked on Oct 25 2012

Answer

Thank you for contacting us and your interest in our products.

Epitomics did offer isotype controls specific for different lots of antibody purchased. This was providing the antibody at the same concentration as the antibody to be tested. We do not currently have this available from our catalogue. We do however have a rabbit monoclonal isotype control. This can be viewed from the following link:

www.abcam.com/ab125938

This is provided at a concentration of 0.2 mg/mL.

I hope this information has been of help. If I can be of any further assistance, please do not hesitate to let me know.

Abcam Scientific Support

Answered on Oct 25 2012

Application

Western blot

Sample

Human Cell lysate - whole cell (HEK293)

Loading amount

200000 cells

Specification

HEK293

Gel Running Conditions

Reduced Denaturing (5% gel)

Blocking step

Milk as blocking agent for 16 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 4°C

Abcam user community

Verified customer

Submitted Feb 02 2010

Application

Western blot

Sample

Human Cell lysate - whole cell (A549 tumour cell line)

Loading amount

50 µg

Specification

A549 tumour cell line

Treatment

100IU bleomycin for 2h

Gel Running Conditions

Reduced Denaturing (3-8% tris acetate gel)

Blocking step

Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 23°C

Abcam user community

Verified customer

Submitted Nov 27 2009

Application

Western blot

Sample

Mouse Tissue lysate - whole (mouse muscle)

Loading amount

100 µg

Specification

mouse muscle

Gel Running Conditions

Reduced Denaturing (6% gel)

Blocking step

Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 25°C

Abcam user community

Verified customer

Submitted Sep 14 2009

Application

Western blot

Sample

Armenian Hamster Tissue lysate - whole (Liver tissue)

Loading amount

50 µg

Specification

Liver tissue

Gel Running Conditions

Reduced Denaturing (6% gel)

Blocking step

Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 25°C

Abcam user community

Verified customer

Submitted Sep 14 2009

Application

Western blot

Sample

African Green Monkey Cell lysate - whole cell (COS-7 cell line)

Loading amount

20 µg

Specification

COS-7 cell line

Gel Running Conditions

Reduced Denaturing (6% gel)

Blocking step

Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 25°C

Abcam user community

Verified customer

Submitted Sep 11 2009

Application

Western blot

Sample

Rat Cell lysate - whole cell (C6 Cell line)

Loading amount

25 µg

Specification

C6 Cell line

Gel Running Conditions

Reduced Denaturing (6% gel)

Blocking step

Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 25°C

Abcam user community

Verified customer

Submitted Jun 11 2008

1-10 of 14 Abreviews or Q&A

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Key features and details

Overview

Product name

Description

Host species

Specificity

Tested Applications & Species

Immunogen

Positive control

General notes

Properties

Form

Storage instructions

Storage buffer

Purity

Clonality

Clone number

Isotype

Research areas

Associated products

Alternative Versions

Compatible Secondaries

Isotype control

Recombinant Protein

Applications

The Abpromise guarantee

Target

Function

Sequence similarities

Post-translationalmodifications

Cellular localization

Database links

Alternative names

Images

Protocols

Datasheets and documents

SDS download

Datasheet download

References (15)

Customer reviews and Q&As

Western blot abreview for Anti-DNA PKcs antibody [Y393]

Immunocytochemistry/ Immunofluorescence abreview for Anti-DNA PKcs antibody [Y393]

Western blot abreview for Anti-DNA PKcs antibody [Y393]

Western blot abreview for Anti-DNA PKcs antibody [Y393]

Western blot abreview for Anti-DNA PKcs antibody [Y393]

Western blot abreview for Anti-DNA PKcs antibody [Y393]

Western blot abreview for Anti-DNA PKcs antibody [Y393]

Western blot abreview for Anti-DNA PKcs antibody [Y393]

Western blot abreview for Anti-DNA PKcs antibody [Y393]

Post-translational
modifications