Product nameAnti-CLLD8/SETDB2 antibody
See all CLLD8/SETDB2 primary antibodies
DescriptionRabbit polyclonal to CLLD8/SETDB2
Tested applicationsSuitable for: WB, IHC-P, ICC/IFmore details
Species reactivityReacts with: Human
- 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 notesOur collaborator on this project has found that the antibody works poorly in WB.
Storage instructionsShipped at 4°C. Upon delivery aliquot and store at -20°C or -80°C. Avoid repeated freeze / thaw cycles.
Storage bufferPreservative: 0.02% Sodium Azide
Constituents: 1% BSA, PBS, pH 7.4
Concentration information loading...
PurityImmunogen affinity purified
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.
|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.|
FunctionHistone 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 specificityUbiquitous. Highest expression in heart, testis and ovary.
Sequence similaritiesBelongs to the histone-lysine methyltransferase family.
Contains 1 MBD (methyl-CpG-binding) domain.
Contains 1 pre-SET domain.
Contains 1 SET domain.
Cellular localizationNucleus. Chromosome.
- Information by UniProt
- C13orf4 antibody
- Chromosome 13 open reading frame 4 antibody
- Chronic lymphocytic leukemia deletion region 8 antibody
Anti-CLLD8/SETDB2 antibody (ab5517) at 1 µg/ml +
Recombinant Human CLLD8/SETDB2 protein (ab169554) at 0.01 µg
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.
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
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) »