Anti-CLLD8/SETDB2 antibody (ab5517)

Immunocytochemistry/ Immunofluorescence - CLLD8/SETDB2 antibody (ab5517)

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 ºC using a 1/50 dilution of antibody ab5517. Cells were then washed 3 times and incubated for 1 hour at 37 ºC 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-transfection. Mouse anti-FLAG antibody was detected with a donkey anti-mouse antibody coupled with Alexa Fluor 555 (Molecular Probes) at a 1:500 dilution. ab5517 was detected using a donkey anti-rabbit antibody coupled with Alexa Fluor 488 at a 1/500 dilution. 

Top: anti-FLAG antibody
Middle: ab5517
Bottom: Merge of anti-FLAG and ab5517 (and DAPI) staining.

Genevieve Fourel, Grenoble

Immunocytochemistry/ Immunofluorescence - CLLD8/SETDB2 antibody (ab5517)

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 ºC for 1 hour. Cells were then washed 3 times and incubated at 37 ºC 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.   

Genevieve Fourel, Grenoble

Western blot - CLLD8/SETDB2 antibody (ab5517)

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

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - CLLD8/SETDB2 antibody (ab5517)

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