Overview

  • Product name
    Anti-DNA Polymerase beta antibody [61]
    See all DNA Polymerase beta primary antibodies
  • Description
    Mouse monoclonal [61] to DNA Polymerase beta
  • Host species
    Mouse
  • Tested applications
    Suitable for: WB, IHC-P, IHC-Fr, ICC/IF, Flow Cytmore details
  • Species reactivity
    Reacts with: Mouse, Human
  • Immunogen

    BALB/C mice were injected with DNA polymerase beta protein.

  • Positive control
    • Testis

Properties

Applications

Our Abpromise guarantee covers the use of ab1831 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 Use at an assay dependent concentration. Predicted molecular weight: 38 kDa.
IHC-P Use at an assay dependent concentration.
IHC-Fr Use at an assay dependent concentration.
ICC/IF Use at an assay dependent concentration. (PMID 20019666).
Flow Cyt Use 1µg for 106 cells.

ab170191 - Mouse monoclonal IgG2a, is suitable for use as an isotype control with this antibody.

 

Target

  • Function
    Repair polymerase. Conducts "gap-filling" DNA synthesis in a stepwise distributive fashion rather than in a processive fashion as for other DNA polymerases. Has a 5'-deoxyribose-5-phosphate lyase (dRP lyase) activity.
  • Sequence similarities
    Belongs to the DNA polymerase type-X family.
  • Domain
    Residues 239-252 form a flexible loop which appears to affect the polymerase fidelity.
  • Post-translational
    modifications
    Methylation by PRMT6 stimulates the polymerase activity by enhancing DNA binding and processivity.
  • Cellular localization
    Nucleus, Cytoplasm
  • Information by UniProt
  • Database links
  • Alternative names
    • DNA directed DNA polymerase beta antibody
    • DNA pol beta antibody
    • DNA polymerase beta antibody
    • DNA polymerase beta subunit antibody
    • DPOLB_HUMAN antibody
    • MGC125976 antibody
    • Pol B antibody
    • Pol beta antibody
    • POLB antibody
    • Polymerase (DNA directed) beta antibody
    see all

Images

  • All lanes : Anti-DNA Polymerase beta antibody [61] (ab1831)

    Lane 1 : Empty vector
    Lane 2 : Wild-type mouse embryonic fibroblasts
    Lane 3 : G231D mutant mouse embryonic fibroblasts
    Lane 4 : T292I mutant mouse embryonic fibroblasts
    Lane 5 : S275N/E295K mutant mouse embryonic fibroblasts

    Predicted band size: 38 kDa



    The normalized level of DNA Polymerase beta is indicated below each lane
  • ab1831 staining human testis by IHC-P.
  • Overlay histogram showing Jurkat cells stained with ab1831 (red line). The cells were fixed with 4% paraformaldehyde and then permeabilized with 0.1% PBS-Tween for 20 min. The cells were then incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions followed by the antibody (ab1831, 1µg/1x106 cells) for 30 min at 22ºC. The secondary antibody used was DyLight® 488 goat anti-mouse IgG (H+L) (ab96879) at 1/500 dilution for 30 min at 22ºC. Isotype control antibody (black line) was mouse IgG1 [ICIGG1] (ab91353, 2µg/1x106 cells) used under the same conditions. Acquisition of >5,000 events was performed.

References

This product has been referenced in:
See all 11 Publications for this product

Customer reviews and Q&As

1-8 of 8 Abreviews or Q&A

Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Western blot
Sample
Human Cell lysate - whole cell (Small Cell Lung Cancer)
Loading amount
5 µg
Specification
Small Cell Lung Cancer
Treatment
POLB over expressed
Gel Running Conditions
Reduced Denaturing (10%)
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 Oct 15 2009

Answer

Thank you for your email. The antibody solution consists of 1X PBS, pH 7.5; 1% BSA; and 0.5 % sodium azide. Please contact us again if you have any additional questions.

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Answer

The storage buffer is PBS, stabilizer is BSA and preservative is Sodium azide. Please contact us again if you have any additional questions.

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Answer

Thank you for contacting us for technical support with ab1831. This antibody has not been tested on mouse samples and we would not predict it to work on mouse tissue samples in immunohistochemical techniques due to the problem of mouse-on-mouse staining whereby the secondary antibody will bind to the whole tissue and not specifically to the protein of interest. Therefore the original staining you got may be non specific. I would recommend running a positive control of human testis in paraffin sections. There are mouse-on-mouse staining kits which help decrease the background staining levels (DAKO kits called ARC kits) and this may help you. I would also recommend permeabilising more by adding 0.1%tritonX100 in the PBS used to dilute the blocking agent and antibodies. Please try without BSA in the blocking buffer as this sometimes gives problems. You do not mention a secondary antibody with biotin conjugation, please make sure the antibody works well with other primaries and that it is incubated 1-2hrs prior to ABC incubation (ABC should be prepared 30min in advance). I hope this information will help you, please do not hesitate to contact us again if you require further assistance,

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Question
Answer

Thank you for your enquiry. This antibody was purified by protein A chromatography, and the concentration is approximately 100 ug/ml. If you have any additional questions, please contact us again.

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Question

BATCH NUMBER 93675 ORDER NUMBER R324750 DESCRIPTION OF THE PROBLEM No staining. I recently had a problem-using ab 1831. When I used for both immunofluorescence and imunohistochemistry I could not get any signal. Before I used yours antibody for both applications and I did not have any problem. Could you help me to solve this big problem for us? I should mention that we do not have a problem using this ab for western. I tried to find out the batch number for the ab 1831, which was working fine for us a year ago. I have already contacted with technical support person and gave him the date of purchasing the working for us AB. It was March 9 2004 and I guess the batch # is 42302. If you have some sample with this batch # could you please exchange with the last one, which did not work for us. SAMPLE mice testis PRIMARY ANTIBODY Immunohistochemistry:ab1831 1:10 dilution in 3% BSA / 1.5% Normal Horse Serum/PBS 16hrs at 4oC Wash step: 3 times 1XPBS Immunofluorescence: ab1831 1:25 dilution in PBTG (1XPBS, 0.2% BSA, 01% Tween20, 0.1% gelatin)/1:200 dil Normal donkey Serum Wash step: 3 times 1XPBS DETECTION METHOD Immunohistochemistry: ABC kit from Vector laboratories-8mklA+8mklB to 0.4ml PBS 30 min pre incubation time and add to the sections for 30 more min. at RT Wash step: 3 times 1XPBS DAB detection: dissolve DAB in 5ml H20 and add UREA/H202 filter and incubate for 5 min at RT onto section Wash step 5 min H20 Hematoxylin for 15 sec, wash once H20 Dehydrate in 70, 95 and 100 % ETOH 5 min each; two times 100% xylene-5 min each mount with: Histological Mounting medium SO-P-15 Immunofluorescence: After wash step stain with DAPI 0.25 mkg/ml for 5 min at RT Wash step 5 min H20 Dry and mount slides using Fluoromount-G +2.5% DABCO POSITIVE AND NEGATIVE CONTROLS USED For immunohistochemistry and immunofluorescence as positive control I used other Abs with the same conditions and they worked perfectly. As negative controls in both applications omitted only primary AB ANTIBODY STORAGE CONDITIONS We always use to aliquot ab1831- DNA polymerase beta in aliquot of 10 mkl and keep them at-80. Working aliquot we use to keep at +4oC no longer than 1 month. FIXATION OF SAMPLE PFA fixed spread prepared from mice testis-immunofluorescence. Formalin fixed and paraffin embedded and section form mice testis for immunohistochemistry. ANTIGEN RETRIEVAL Immunohistochemistry: 10mM citric acid pH 6.0 15 min 90-95oC. Immunofluorescence: quenched with 0.1% glycine/PBS for 10 min PERMEABILIZATION STEP Immunohistochemistry:0.1 % H202 for 20 min at RT and 0.05 % saponin in d2 H20 for 30 min Immunofluorescence: 0.5% Tritonx100-30 min at RT BLOCKING CONDITIONS Immunohistochemistry: 3% BSA / 1.5% Normal Horse Serum/PBS for 1 hr at RT Immunofluorescence: PBTG (1XPBS, 0.2% BSA, 01% Tween20, 0.1% gelatin)/1:200 dil Normal donkey Serum SECONDARY ANTIBODY Immunohistochemistry: Vector Laboratories, Biotinylated Anti Mouse IgG (H+L) made in horse Dilution 1:200 in 3% BSA / 1.5% Normal Horse Serum/PBS for 1hour at RT Wash step: 3 times 1XPBS Immunofluorescence: Jackson ImmunoResearch Laboratories anti Mouse IgG (H+L) FITC conjugated made in donkey. Dilution 1:100 in PBTG (1XPBS, 0.2% BSA, 01% Tween20, 0.1% gelatin)/1:200 diluted Normal donkey Serum for 1hour at RT Wash step: 3 times 1XPBS HOW MANY TIMES HAVE YOU TRIED THE APPLICATION? 5 HAVE YOU RUN A "NO PRIMARY" CONTROL? Yes DO YOU OBTAIN THE SAME RESULTS EVERY TIME? Yes WHAT STEPS HAVE YOU ALTERED? Checked different dilutions bud did not get results even with 1:10 dilution for both applications.

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Answer

I'm sorry to hear you are having problems with ab1831 in IHC. The fact that the antibody still works in WB suggests that the antibody is fine but that there is a component in the IHC protocol which is damaged, there could potentially be many "ingredients" used in the IHC protocol which are damaged and prevent good staining. We have not tested the antibody on mouse tissue and therefore cannot guarantee that it will work in mouse. Since you purchased the antibody in march 2004, it is more than a year old, and therefore it is also possible that it has been damaged during storage in your freezer, you also say "working aliquot we use to keep at +4oC no longer than 1 month", I would recommend trying fresh aliquots in case those were damaged during storage at 4C.

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Question

The batch that worked was purchased on the beginning of Aril, 2004. Basically I treat mono-layer of cultured cell with SDS-loading buffer, boil it for 5 minutes before I load it into PAGE. These cells were either induced or not for the expression of a DNA polymerase beta mutant. With a good batch of anti-pol beta monoclonal antibody, I'd expect to see a single band on non-induced samples and two bands on induced samples. I hope this would help. The Reference number is: 56538 (PON: R3012207). The date of purchase is Oct.13, 2004. The problem with this batch of antibody is that there's no signal on the membrane with high dilution. Background is minimal so what I got is a blank blot. I don't have the batch number of the antibody that worked since I don't have the original tube. My samples are cellular extract. I had no problems with the batch that worked. I had tried this batch (60820) many times with the exact protocol. My finding is that if I don't dilute it to 1: 200, there will be no signals. So I am pretty sure this batch of antibody has lower titer compared with the older batch (dilution: 1:1000). Considering those, I think we should get another batch of this antibody without charge to test until we can get good signal. Thanks a lot for your time! I recently ordered anti-DNA polymerase beta-ab1831, batch 60820 from your company. The same antibody we ordered before used to work well for Western blot with a dilution of 1: 1000. But this batch never worked at this dilution until it's increased at least to 1: 200. So can you help me find out what's wrong? Thanks a lot!

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Answer

Thank you for the information. We have not received any other complaints regarding this antibody and since the previous vial of ab1831 worked for you, it sounds like perhaps there is a problem with the latest vial that you received. I'm sending you a replacement vial free of charge and for your record it is being shipped to your attention on Abcam order# 62985. You should receive this tomorrow. Please let me know if you have any additional questions and please also let me know how the replacement works out for you.

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