Overview

  • Product name
    Anti-DNA polymerase eta antibody
    See all DNA polymerase eta primary antibodies
  • Description
    Rabbit polyclonal to DNA polymerase eta
  • Specificity
    ab17725 specifically recognizes a band at ~80 kDa, the reported size of DNA polymerase eta, in MRC5 cells. This band is not seen in XP30RO cells, which lack endogenous DNA polymerase eta.
  • Tested applications
    Suitable for: WB, ICC/IFmore details
  • Species reactivity
    Reacts with: Human
  • Immunogen

    Synthetic peptide conjugated to KLH derived from within residues 650 to the C-terminus of Human DNA polymerase eta.

    (Peptide available as ab19077.)

  • Positive control
    • This antibody gave a positive signal in: (WB) HeLa

Properties

  • Form
    Liquid
  • Storage instructions
    Shipped at 4°C. Store at +4°C short term (1-2 weeks). Upon delivery aliquot. Store at -20°C or -80°C. Avoid freeze / thaw cycle.
  • Storage buffer
    Preservative: 0.02% Sodium Azide
    Constituents: 1% BSA, PBS, pH 7.4
  • Concentration information loading...
  • Purity
    Immunogen affinity purified
  • Clonality
    Polyclonal
  • Isotype
    IgG
  • Research areas

Applications

Our Abpromise guarantee covers the use of ab17725 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
WB 1/400 - 1/2000. Detects a band of approximately 79-85 kDa (predicted molecular weight: 79 kDa).
ICC/IF Use at an assay dependent concentration. PubMed: 25421715

Target

  • Function
    DNA polymerase specifically involved in DNA repair. Plays an important role in translesion synthesis, where the normal high fidelity DNA polymerases cannot proceed and DNA synthesis stalls. Plays an important role in the repair of UV-induced pyrimidine dimers. Depending on the context, it inserts the correct base, but causes frequent base transitions and transversions. May play a role in hypermutation at immunoglobulin genes. Forms a Schiff base with 5'-deoxyribose phosphate at abasic sites, but does not have lyase activity. Targets POLI to replication foci.
  • Involvement in disease
    Defects in POLH are the cause of xeroderma pigmentosum variant type (XPV) [MIM:278750]; also designated as XP-V. Xeroderma pigmentosum (XP) is an autosomal recessive disease due to deficient nucleotide excision repair. It is characterized by hypersensitivity of the skin to sunlight, followed by high incidence of skin cancer and frequent neurologic abnormalities. XPV shows normal nucleotide excision repair, but an exaggerated delay in recovery of replicative DNA synthesis. Most XPV patients do not develop clinical symptoms and skin neoplasias until a later age. Clinical manifestations are limited to photo-induced deterioration of the skin and eyes.
  • Sequence similarities
    Belongs to the DNA polymerase type-Y family.
    Contains 1 umuC domain.
  • Domain
    The catalytic core consists of fingers, palm and thumb subdomains, but the fingers and thumb subdomains are much smaller than in high-fidelity polymerases; residues from five sequence motifs of the Y-family cluster around an active site cleft that can accommodate DNA and nucleotide substrates with relaxed geometric constraints, with consequently higher rates of misincorporation and low processivity.
  • Cellular localization
    Nucleus. Accumulates at replication forks after DNA damage.
  • Information by UniProt
  • Database links
  • Alternative names
    • DNA polymerase eta antibody
    • FLJ16395 antibody
    • FLJ21978 antibody
    • Polh antibody
    • POLH_HUMAN antibody
    • polymerase DNA directed eta antibody
    • RAD30 antibody
    • RAD30 homolog A antibody
    • RAD30A antibody
    • Xeroderma pigmentosum variant type protein antibody
    • XP V antibody
    • XPV antibody
    see all

Anti-DNA polymerase eta antibody images



  • Performed under reducing conditions.

    Predicted band size : 79 kDa
    Observed band size : 90 kDa (why is the actual band size different from the predicted?)

    ane 1: Wild-type HAP1 cell lysate (20 µg)
    Lane 2: DNA poly eta knockout HAP1 cell lysate (20 µg)
    Lane 3: XP30RO cell lysate (20 µg)
    Lane 4: MRC5 cell lysate (20 µg)
    Lanes 1 - 4: Merged signal (red and green). Green - ab17725 observed at 90 kDa. Red - loading control, ab8245, observed at 37 kDa.

    ab17725 was shown to recognize DNA pol eta when DNA pol eta knockout samples were used, along with additional cross-reactive bands. Wild-type and DNA pol eta knockout samples were subjected to SDS-PAGE. ab17725 1ug/ml and ab8245 (loading control to GAPDH) at a dilution of 1/10 000 were 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 1/10000 dilution for 1 hour at room temperature before imaging.

    XP30RO and MRC5 cell lysates were provide and a gift from Prof Catherine Green (Wellcome Trust Centre for Human Genetics, Univeristy of Oxford)

     

  • Lanes 1, 2, 5 & 6 : Anti-DNA polymerase eta antibody (ab17725) at 1/400 dilution
    Lanes 3 - 4 : Anti-DNA polymerase eta antibody (ab17725) at 1/2000 dilution

    Lane 1 : XP30RO cell lysate (lacking endogenous DNA polymerase eta)
    Lane 2 : MRC5 cell lysate
    Lane 3 : XP30RO cell lysate (lacking endogenous DNA polymerase eta)
    Lane 4 : MRC5 cell lysate
    Lane 5 : XP30RO cell lysate(lacking endogenous DNA polymerase eta) with Human DNA polymerase eta peptide (ab19077) at 5 µl
    Lane 6 : MRC5 cell lysate with Human DNA polymerase eta peptide (ab19077) at 5 µl

    Secondary
    Goat anti-rabbit HRP at 1/5000 dilution
    Developed using the ECL technique

    Performed under reducing conditions.

    Predicted band size : 79 kDa
    Observed band size : 79 kDa


    Exposure time : 20 minutes

    60% confluent 6cm dish of each cell type was washed in PBS then scraped into 150µl of SDS-PAGE buffer, boiled for 5 minutes then sonicated for 10 minutes. 20µl of each sample was loaded in triplicate on a 6.5% polyacrylamide denaturing gel, then transferred to nitrocellulose. The membrane was blocked in 5% non-fat milk in PBS plus 0.1% Tween20 for 30 minutes. The primary antibody, ab17725, was incubated with the membrane in PBS plus 0.1% Tween20 and 5% milk overnight. Where the immunizing peptide was used, 5µl of the peptide was incubated with 5ul of antibody for ten minutes prior to dilution in 2ml of 5% milk in PBS plus 0.1% Tween20, which was then incubated with the blocked membrane overnight.

References for Anti-DNA polymerase eta antibody (ab17725)

This product has been referenced in:
  • Sekimoto T  et al. Both high-fidelity replicative and low-fidelity Y-family polymerases are involved in DNA rereplication. Mol Cell Biol 35:699-715 (2015). Read more (PubMed: 25487575) »
  • Fattah FJ  et al. The transcription factor TFII-I promotes DNA translesion synthesis and genomic stability. PLoS Genet 10:e1004419 (2014). Human . Read more (PubMed: 24922507) »

See all 21 Publications for this product

Product Wall

Application
Western blot
Loading amount
35 µg
Gel Running Conditions
Reduced Denaturing (4-12% Bis-Tris)
Sample
Mouse Cell lysate - whole cell (HEK293, HCT116, U2OS and Mouse Embryonic Fibroblas)
Specification
HEK293, HCT116, U2OS and Mouse Embryonic Fibroblas
Blocking step
Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 21°C
Username

Abcam user community

Verified customer

Submitted Dec 23 2013

Application
Western blot
Loading amount
35 µg
Gel Running Conditions
Reduced Denaturing (4-12% Bis-Tris)
Sample
Human Cell lysate - whole cell (HEK293, HCT116, U2OS and Mouse Embryonic Fibroblas)
Specification
HEK293, HCT116, U2OS and Mouse Embryonic Fibroblas
Blocking step
Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 21°C
Username

Abcam user community

Verified customer

Submitted Dec 23 2013

Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Immunocytochemistry/ Immunofluorescence
Blocking step
BSA as blocking agent for 2 hour(s) and 0 minute(s) · Concentration: 2%
Sample
Human Cell (U2OS)
Specification
U2OS
Permeabilization
Yes - 0.5% triton
Fixative
Paraformaldehyde
Username

Dr. Christophe Lachaud

Verified customer

Submitted Jul 31 2013

Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Western blot
Loading amount
25 µg
Gel Running Conditions
Reduced Denaturing
Sample
Human Cell lysate - whole cell (293 cells)
Specification
293 cells
Blocking step
Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 22°C
Username

Abcam user community

Verified customer

Submitted Jul 24 2013

Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Immunocytochemistry/ Immunofluorescence
Sample
Human Cell (U2OS cells)
Specification
U2OS cells
Fixative
Formaldehyde
Permeabilization
Yes - np40
Blocking step
BSA as blocking agent for 2 hour(s) and 0 minute(s) · Concentration: 3% · Temperature: rt°C
Username

Abcam user community

Verified customer

Submitted Feb 15 2013

Thank you for your reply.

Indeed it looks like the antibody should have worked under these conditions. We have a different lot availible, and I am happy to provide this free of charge for your customer as they are covered by our Abpromise. Pl...

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Thank you for taking time to complete our questionnaire. I am sorry to hear that this antibody is not providing satisfactory results for your customer.



Having reviewed this case, I would appreciate if you can confirm some further...

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Ma collègue m'a transféré votre cas.

Concernant ab17725, nous n’avons que lot GR52626-1 en stock. Nous avons un nouveau lot en production qui devrait être disponible dans environ 2 mois s’il passe nos te...

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I am sorry to hear you are experiencing difficulties with one of our products. We take product complaints very seriously, and investigate every product that we feel may not be performing correctly.
I am attaching ...

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Thank you again for your patience with this.

We are currently transferringtwo vials from our lab stock to our distribution office, and they will be sent to you from there on the free of charge order # ***. I expect this shipment to arrive nex...

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