Overview

  • Product name
    Anti-CLLD8/SETDB2 antibody
    See all CLLD8/SETDB2 primary antibodies
  • Description
    Rabbit polyclonal to CLLD8/SETDB2
  • Host species
    Rabbit
  • Tested applications
    Suitable for: WB, IHC-P, ICC/IFmore details
  • Species reactivity
    Reacts with: Human
  • Immunogen

    Synthetic peptide corresponding to Human CLLD8/SETDB2 aa 550-650 conjugated to keyhole limpet haemocyanin.
    Database link: Q96T68
    (Peptide available as ab24398)

  • Positive control
    • This antibody gave a positive signal when tested against Sf21 cells infected by baculovirus expressing CLLD8/SETDB2 This antibody gave a positive result when used in the following formaldehyde fixed cell lines: MCF-7.
  • General notes
    Our collaborator on this project has found that the antibody works poorly in WB.

Properties

Applications

Our Abpromise guarantee covers the use of ab5517 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. PubMed: 20826732
IHC-P 1/50. Perform heat mediated antigen retrieval via the pressure cooker method before commencing with IHC staining protocol.
ICC/IF Use at an assay dependent concentration.

Target

  • Function
    Histone methyltransferase involved in left-right axis specification in early development and mitosis. Specifically trimethylates 'Lys-9' of histone H3 (H3K9me3). H3K9me3 is a specific tag for epigenetic transcriptional repression that recruits HP1 (CBX1, CBX3 and/or CBX5) proteins to methylated histones. Contributes to H3K9me3 in both the interspersed repetitive elements and centromere-associated repeats. Plays a role in chromosome condensation and segregation during mitosis.
  • Tissue specificity
    Ubiquitous. Highest expression in heart, testis and ovary.
  • Sequence similarities
    Belongs to the histone-lysine methyltransferase family.
    Contains 1 MBD (methyl-CpG-binding) domain.
    Contains 1 pre-SET domain.
    Contains 1 SET domain.
  • Cellular localization
    Nucleus. Chromosome.
  • Information by UniProt
  • Database links
  • Alternative names
    • C13orf4 antibody
    • Chromosome 13 open reading frame 4 antibody
    • Chronic lymphocytic leukemia deletion region 8 antibody
    • Chronic lymphocytic leukemia deletion region gene 8 antibody
    • Chronic lymphocytic leukemia deletion region gene 8 protein antibody
    • Clld8 antibody
    • CLLL8 antibody
    • Gm293 antibody
    • H3-K9-HMTase antibody
    • Histone H3-K9 methyltransferase antibody
    • Histone-lysine N-methyltransferase SETDB2 antibody
    • KMT1F antibody
    • Lysine N-methyltransferase 1F antibody
    • Probable histone-lysine N-methyltransferase H3 lysine-9 specific antibody
    • SEB2 antibody
    • SET domain bifurcated 2 antibody
    • SET domain protein, bifurcated, 2 antibody
    • SETB2_HUMAN antibody
    • Setdb2 antibody
    see all

Images

  • Anti-CLLD8/SETDB2 antibody (ab5517) at 1 µg/ml + Recombinant Human CLLD8/SETDB2 protein (ab169554) at 0.01 µg

    Secondary
    Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/10000 dilution

    Developed using the ECL technique.

    Performed under reducing conditions.

    Additional bands at: 120 kDa (possible tagged protein)


    Exposure time: 10 seconds


    ab5517 recognises a band corresponding to the tagged CLLD8/SETB2 at approximately 120 kDa.

     

    Abcam recommends using milk as the blocking agent. Abcam welcomes customer feedback and would appreciate any comments regarding this product and the data presented above.

  • ICC/IF image of ab5517 stained MCF-7 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 ab5517 at 5ug/ml overnight at +4°C. The secondary antibody (green) was DyLight® 488 Goat anti Rabbit (ab96899) IgG (H+L) used at a 1/250 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.

  • Left: Endogenous CLLD8

    Detection using indirect fluorescence of the signal corresponding to endogenous CLLD8 in 293T cells. Cells fixed using 4% formaldehyde, blocked with PBS containing 3% milk and 0.5% Triton X-100, incubated for 1 hour at 37 °C using a 1/50 dilution of antibody ab5517. Cells were then washed 3 times and incubated for 1 hour at 37 °C with a goat anti-rabbit secondary antibody (1/500 dilution) coupled to Alexa Fluor 555 (Molecular Probes). Following 3 more washes, cells were stained with DAPI and mounted with Vectashield.

    Top: DAPI
    Bottom: ab5517


    Right: Overexpressed CLLD8

    Detection using indirect fluorescence of the signal corresponding to staining with anti-FLAG mouse antibody (top) and an antibody (ab5517) against CLLD8 (middle) in 293T cells. 293T cells were transfected with vectors for overexpression of flagged CLLD8 using Lipofectamine 2000 transfection procedure (Invitrogen). Cells were fixed 48 hours post-tra

  • Detection using indirect fluorescence of the signal corresponding to endogenous CLLD8 in 293T cells. Using Lipofectamine 2000 (Invitrogen), cells were transfected with either a control shRNA directed against luciferase (left, SiRluc) or a specific siRNA directed against CLLD8 (right). 48 hours post-transfection, cells were fixed, blocked, and incubated with a 1:50 dilution of ab5517 at 37 °C for 1 hour. Cells were then washed 3 times and incubated at 37 °C for 1 hour with a 1/500 dilution of goat anti-rabbit antibody coupled with Alexa Fluor 555 (Molecular Probes). Following 3 further washes cells were stained with DAPI and mounted with Vectashield.

    Antibody signals were extinguished when a specific RNAi was used. The few cells which retained a signal were presumably not transfected.   

     

  • All lanes : Anti-CLLD8/SETDB2 antibody (ab5517) at 1 µg/ml

    Lane 1 : Sf21 cells infected by baculovirus containing FLAG-CLLD8 gene at 10 µg
    Lane 2 : Sf21 cells infected by baculovirus containing FLAG-CLLD8 gene at 20 µg
    Lane 3 : non-infected Sf21 cells at 10 µg
    Lane 4 : non-infected Sf21 cells at 20 µg
    Lane 5 : Sf21 infected by baculovirus containing FLAG-CLLD8 gene at 10 µg with Human CLLD8/SETDB2 peptide (ab24398) at 1 µg/ml
    Lane 6 : Sf21 infected by baculovirus containing FLAG-CLLD8 gene at 20 µg with Human CLLD8/SETDB2 peptide (ab24398) at 1 µg/ml
    Lane 7 : non-infected Sf21 cells at 10 µg with Human CLLD8/SETDB2 peptide (ab24398) at 1 µg/ml
    Lane 8 : non-infected Sf21 cells at 20 µg with Human CLLD8/SETDB2 peptide (ab24398) at 1 µg/ml

    Observed band size: 110 kDa
    why is the actual band size different from the predicted?
    Additional bands at: 22 kDa (possible non-specific binding), 90 kDa (possible non-specific binding)



    ab5517 recognises a band corresponding to FLAG-tagged CLLD8/SETB2 at approximately 110 kDa in Sf21 cells infected by baculovirus expressing CLLD8/SETDB2 (lanes1-2). This is larger than the predicated band size (81 kDa) due to the addition of the FLAG tag.

    ab5517 does not detect a band in uninfected sf21 cells (lanes3-4) or following blocking studies using the immunizing peptide (lanes5-6).

    There appears to be a ladder of proteins recognised by ab5517 (lanes1-2), which could be attributed to degradation products.
  • Image courtesy of Human Protein Atlas
    ab5517 staining CLLD8 in human colon tissue and showing a distinct and strong staining of the nucleus of the glandular cells. Paraffin embedded human colon was incubated with ab5517 (1/50) for 30 minutes at room temperature. Antigen retrieval was performed by heat induction in citrate buffer pH6. ab5517 was tested in a tissue microarray (TMA) containing a wide range of normal and cancer tissues as well as a cell microarray consisting of a range of commonly used, well characterised human cell lines. Further results for this antibody can be found at www.proteinatlas.org

References

This product has been referenced in:
  • Nishikawaji T  et al. Oncogenic roles of the SETDB2 histone methyltransferase in gastric cancer. Oncotarget 7:67251-67265 (2016). IHC . Read more (PubMed: 27572307) »
  • Hogarth CA  et al. Identification and expression of potential regulators of the mammalian mitotic-to-meiotic transition. Biol Reprod 84:34-42 (2011). WB, IHC-P ; Mouse . Read more (PubMed: 20826732) »
See all 3 Publications for this product

Customer reviews and Q&As

1-2 of 2 Abreviews or Q&A

Answer

Thank you for your inquiry. I BLASTed the immunogen sequence of ab5517 against mouse and it only has 55% homology.  We generally say that anything with a greater than 85% homology would be expected to cross-react, so this is quite low.  This antibody probably wouldn't work with mouse samples. However, we do have a goat polyclonal anti-CLLD8/SETDB2 antibody that is predicted to cross-react with mouse, ab13712.  This immunogen is 100% homologous with mouse. However, since we have not tested ab13712 in mouse, we would not be able to guarantee that it would work. https://www.abcam.com/CLLD8-SETDB2-antibody-ab13712.html But, I can offer a discount off a future purchase if you buy ab13712 now, test it in mouse 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 ab13712 in mouse.  I will then send a discount code. This code must be issued before purchasing ab13712 so please wait for my reply before ordering. 2. Purchase ab13712 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 any primary antibody ordered 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 ab13712 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

I'm very sorry to hear you are having problems with ab5517 and ab13712. You do not mention adding loading buffer to your samples prior to boiling, please make sure you add loading buffer containing SDS, DTT, beta-mercaptoethanol and bromphenol blue. Please also make sure you keep samples on ice until boiling and that protease inhibitors are added freshly. You do not mention how long you block the membrane for (and what temperature), I recommend trying 5%BSA in TBST for 1 hr, then rinsing a few seconds in TBST and incubating both primary and secondary antibodies in TBST. The dilution recommended for ab5517 is 1:5000 and for ab13712 is ug/ml but you may need to dilute more, I hope these suggestions help, please do not hesitate to contact us if you need further assistance,

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