Overview

  • Product name

    Anti-DDB2 antibody [2246C4a]
    See all DDB2 primary antibodies
  • Description

    Mouse monoclonal [2246C4a] to DDB2
  • Host species

    Mouse
  • Tested applications

    Suitable for: IHC-P, ICC/IF, WB, Dot blot, Flow Cytmore details
  • Species reactivity

    Reacts with: Human
  • Immunogen

    Recombinant fragment (N-terminal) Human.

  • Positive control

    • HeLa whole cell lysate

Properties

Applications

Our Abpromise guarantee covers the use of ab51017 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
IHC-P Use a concentration of 5 µg/ml. Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.
ICC/IF Use a concentration of 10 µg/ml.
WB 1/50. Detects a band of approximately 45 kDa (predicted molecular weight: 48 kDa).
Dot blot Use at an assay dependent concentration.
Flow Cyt Use 1µg for 106 cells.

ab170190 - Mouse monoclonal IgG1, is suitable for use as an isotype control with this antibody.

 

Target

  • Function

    Required for DNA repair. Binds to DDB1 to form the UV-damaged DNA-binding protein complex (the UV-DDB complex). The UV-DDB complex may recognize UV-induced DNA damage and recruit proteins of the nucleotide excision repair pathway (the NER pathway) to initiate DNA repair. The UV-DDB complex preferentially binds to cyclobutane pyrimidine dimers (CPD), 6-4 photoproducts (6-4 PP), apurinic sites and short mismatches. Also appears to function as the substrate recognition module for the DCX (DDB1-CUL4-X-box) E3 ubiquitin-protein ligase complex DDB1-CUL4-ROC1 (also known as CUL4-DDB-ROC1 and CUL4-DDB-RBX1). The DDB1-CUL4-ROC1 complex may ubiquitinate histone H2A, histone H3 and histone H4 at sites of UV-induced DNA damage. The ubiquitination of histones may facilitate their removal from the nucleosome and promote subsequent DNA repair. The DDB1-CUL4-ROC1 complex also ubiquitinates XPC, which may enhance DNA-binding by XPC and promote NER. Isoform D1 and isoform D2 inhibit UV-damaged DNA repair.
  • Tissue specificity

    Ubiquitously expressed; with highest levels in corneal endothelium and lowest levels in brain. Isoform D1 is highly expressed in brain and heart. Isoform D2, isoform D3 and isoform D4 are weakly expressed.
  • Pathway

    Protein modification; protein ubiquitination.
  • Involvement in disease

    Defects in DDB2 are a cause of xeroderma pigmentosum complementation group E (XP-E) [MIM:278740]; also known as xeroderma pigmentosum V (XP5). XP-E is a rare human autosomal recessive disease characterized by solar sensitivity, high predisposition for developing cancers on areas exposed to sunlight and, in some cases, neurological abnormalities.
  • Sequence similarities

    Belongs to the WD repeat DDB2/WDR76 family.
    Contains 5 WD repeats.
  • Domain

    The DWD box is required for interaction with DDB1.
  • Post-translational
    modifications

    Phosphorylation by ABL1 negatively regulate UV-DDB activity.
    Ubiquitinated by CUL4A in response to UV irradiation. Ubiquitination appears to both impair DNA-binding and promotes ubiquitin-dependent proteolysis. Degradation of DDB2 at sites of DNA damage may be a prerequisite for their recognition by XPC and subsequent repair. CUL4A-mediated degradation appears to be promoted by ABL1.
  • Cellular localization

    Nucleus. Accumulates at sites of DNA damage following UV irradiation.
  • Information by UniProt
  • Database links

  • Alternative names

    • damage-specific DNA binding protein 2 antibody
    • Damage-specific DNA-binding protein 2 antibody
    • DDB p48 subunit antibody
    • Ddb2 antibody
    • DDB2_HUMAN antibody
    • DDBb antibody
    • DNA damage-binding protein 2 antibody
    • UV-damaged DNA-binding protein 2 antibody
    • UV-DDB 2 antibody
    • Xeroderma pigmentosum group E protei antibody
    see all

Images

  • Anti-DDB2 antibody [2246C4a] (ab51017) at 1/50 dilution + HeLa whole cell lysate at 50 µg

    Secondary
    Mouse IgG antibody at 1/2500 dilution

    Developed using the ECL technique.

    Predicted band size: 48 kDa
    Observed band size: 45 kDa
    why is the actual band size different from the predicted?

  • IHC image of ab51017 staining in human normal cervical carcinoma formalin fixed paraffin embedded tissue section, performed on a Leica BondTM system using the standard protocol F. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20 mins. The section was then incubated with ab51017, 5µg/ml, for 15 mins at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX.

    For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.

  • ICC/IF image of ab51017 stained HeLa cells. The cells were 4% formaldehyde fixed (10 min) and then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1h to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab51017, 10µg/ml) overnight at +4°C. The secondary antibody (green) was Alexa Fluor® 488 goat anti-mouse IgG (H+L) used at a 1/1000 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (red) at a 1/200 dilution for 1h. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43µM.

  • Overlay histogram showing HeLa cells stained with ab51017 (red line). The cells were fixed with 80% methanol (5 min) 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. The cells were then incubated with the antibody (ab51017, 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:

  • Zhang X  et al. The CtBP1-HDAC1/2-IRF1 transcriptional complex represses the expression of the long noncoding RNA GAS5 in human osteosarcoma cells. Int J Biol Sci 15:1460-1471 (2019). Read more (PubMed: 31337976) »
  • Piquet S  et al. The Histone Chaperone FACT Coordinates H2A.X-Dependent Signaling and Repair of DNA Damage. Mol Cell 72:888-901.e7 (2018). Read more (PubMed: 30344095) »
See all 13 Publications for this product

Customer reviews and Q&As

1-4 of 4 Abreviews or Q&A

Application
Immunocytochemistry/ Immunofluorescence
Sample
Human Cell (Fibroblasts)
Specification
Fibroblasts
Fixative
Paraformaldehyde
Permeabilization
Yes - 0.5% Triton X-100
Blocking step
Serum as blocking agent for 16 hour(s) and 0 minute(s) · Concentration: 20% · Temperature: 4°C

Dr. Qien Wang

Verified customer

Submitted Jul 11 2012

Answer

Thank you very much for your interest in ab51017.

In general the testing discount program is meant for standard applications that have not been tested with a certain antibody yet. So ab51017 would not be eligible in IF staining since this is already tested.

Since you are offering an image of thisantibody I am happy to make a special exception and provide you with a testing discount code.

DISCOUNT CODE: 100%ABR-00000

Expiration date: 04. October 2012

I am very pleased to hear you used ab51017 inIF and send us an image of your results.

This code will give you 1 free primary antibody before the expiration date. To redeem this offer, please submit an Abreview with the image for IF and include this code in the “Additional Comments” section so we know the Abreview is for this promotion. Please remember that submission of the Abreview is sufficient for the discount code to become active.
For more information on how to submit an Abreview, please visit the site: www.abcam.com/Abreviews.

The code will be active once the Abreview with the image 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. Remember if the product does not work you will be covered by our Abpromise guarantee and eligible for a full refund or replacement.

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.

Read More
Application
Western blot
Sample
Human Cell lysate - nuclear (GM00637 fibroblast cell line)
Loading amount
50 µg
Specification
GM00637 fibroblast cell line
Gel Running Conditions
Reduced Denaturing (10 %)
Blocking step
Blocking buffer from another company as blocking agent for 30 minute(s) · Concentration: 5µg/mL · Temperature: 22°C

Mrs. Annika Schäfer

Verified customer

Submitted Feb 10 2009

Answer

Thank you for your enquiry. I am sorry to hear that you are experiencing difficulties with this product ab51017 in western blot. Often it is possible to make suggestions that help resolve problems. We will happily offer technical support and in the event that a product is not functioning in the applications cited on the product data sheet (and the problem has been reported within 120 days of purchase), and if it appears that the antibody is at fault, a replacement/credit note/refund will be offered. After looking at the protocols you used, I am surprised that even 50ug of protein, you still did not manage to get any bands. However, it is very hard for me to determine the cause without more details of your WB protocol. I would therefore appreciate if you can please clarify the following items. According to the datasheet, this product has a recommended dilution of 1:50. Please try this dilution as previous tests have shown that a higher than normal range is needed for this product to work. Was the lysis buffer you used as strong as RIPA buffer? Please try RIPA buffer as it is the strongest and will ensure that you get all the protein of interest out. Did you use reduce and denaturing conditions? Please reduce and denature the sample at 95oC for 10 minutes in buffer containing SDS and mercaptoethanol. This will ensure that the protein is in the correct conformation to run at the correct molecular weight and be detected by the antibody. Most customers who use milk as a blocking agent tend to have problems detecting bands. At Abcam, almost all of our products have been tested with 5% BSA. Sometimes, a different blocking buffer might cause cross reaction between the blocking agent and antibodies, therefore causing weak bands or no bands at all. I would recommend trying 5% BSA, if you have not already done so. Also, cross reaction between the antibodies with the diluent could also cause the lack of signals. We would normally use 1% BSA to dilute the primary and secondary antibodies. You can also use PBST only. Can you confirm that the second antibody you used also managed to produce good results with other primary antibodies? In some cases, the problem may be with the secondary antibody not working. Could the no signal you are experiencing be due to poor transfer of protein to membrane? Did you check the transfer efficiency? This can be simply done with a reversible stain such as Ponceau S. I am sorry for the string of questions but I hope the above recommendations may already help you. If you have already tried the above suggestions and still experience problems, please do not hesitate to contact me with details of your order shipping address/purchasing agent information. Also, please advice me on how you would like to proceed with your enquiry, so that I can immediately arrange for a replacement or refund to you.

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