Anti-Sumo 2 + Sumo 3 抗体 (ab3742)

ab3742 staining Sumo 2 + Sumo 3 in HepG2 cells. The cells were fixed with 4% paraformaldehyde (10 min), permeabilized with 0.1% PBS-Triton X-100 for 5 minutes and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1%PBS-Tween for 1h. The cells were then incubated overnight at 4°C with ab3742 at 1µg/ml and ab7291, Mouse monoclonal [DM1A] to alpha Tubulin - Loading Control. Cells were then incubated with ab150081, Goat polyclonal Secondary Antibody to Rabbit IgG - H&L (Alexa Fluor® 488), pre-adsorbed at 1/1000 dilution (shown in green) and ab150120, Goat polyclonal Secondary Antibody to Mouse IgG - H&L (Alexa Fluor® 594), pre-adsorbed at 1/1000 dilution (shown in pseudocolour red). Nuclear DNA was labelled with DAPI (shown in blue).

Image was acquired with a high-content analyser (Operetta CLS, Perkin Elmer) and a maximum intensity projection of confocal sections is shown.

Left-hand images show uninfected cells and the co-localization of SUMO-2/3 with PML (red) in control (upper rows of each block of 4 images) and PML depleted (low rows of each block of 4 images) HepaRG cells. Right-hand images show typical examples of recruitment of the indicated proteins to sites associated with HSV-1 genomes (ICP4; red) in cells at the edges of ICP0 null mutant (ΔICP0) plaques in control and PML depleted HepaRG cells. Scale bars indicate 5 µm.

Cells on glass coverslips were fixed with 1.5% formaldehyde in PBS containing 2% sucrose then treated with 0.5% Nonidet P40 substitute in PBS/10% sucrose. SUMO-2/3 was detected with ab3742. An Alexa-conjugated anti-rabbit IgG was used as the secondary antibody.

Lanes 1 & 3: Sumo 2+3 antibody (ab3742) at 1/500 dilution
Lanes 2 & 4: Sumo 2+3 antibody (ab3742) at 1/1000 dilution
Lanes 1-4: HeLa nuclear extract at 20 ug

Lane 1: as above
Lane 2: as above
Lane 3: Sumo 2+3 peptide (ab13760) at 1 ug/ml
Lane 4: Sumo 2+3 peptide (ab13760) at 1 ug/ml

Secondary
Goat polyclonal to Rabbit IgG H&L (HRP) (ab6721) at 1/2000 dilution developed using the ECL technique

Performed under reducing conditions.
 
Exposure time: 1 minute

Predicted band sizes : 11.6 & 10.

Image courtesy of Human Protein Atlas

ab3742 staining Sumo 2 + 3 in Human skin. The  paraffin embedded human skin tissue was incubated with ab3742 (1/800 dilution) for 30 mins at room temperature. Antigen retrieval was performed by heat induction in citrate buffer pH 6. Ab3742 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

Immunofluorescent imaging of human cells (U2OS) with ab3742 confirms the specificity of this antibody.  Antibody signal is localised exclusively to the nucleus, with diffuse background staining of the nucleoplasm.  Intense foci of staining are also evident, corresponding to SUMO-2/3 accumulation in nuclear subdomains such as the PML body.  This image is in exact agreement with several published reports (see for example Saitoh H et al.). 

IF was performed with a standard paraformaldehyde technique (fixed in PBS buffered PFH 4% for 5 minutes, permeabilised with 0.5% triton-PBS for 5 minutes, blocked with 5% milk / 0.2% tween for one hour.  Primary antibody used at 1/100 in 5% milk / 0.2% TWEEN for one hour, secondary antibody for 30 minutes.  All blocking and incubation steps carried out at 37 degrees. Nuclei are stained with Hoechst stain.

ab3742 staining Sumo 2+3 in Xenopus laevis oocyte cell by ICC/IF (Immunocytochemistry/immunofluorescence). Cells were fixed with formaldehyde, permeabilized with NP-40 0.1% in PBS and blocked with 3% BSA for 60 minutes at 23°C. Samples were incubated with primary antibody (1/250 in PBS + 3% BSA) for 1 hour at 23°C. An undiluted Alexa Fluor®488-conjugated Goat anti-rabbit polyclonal was used as the secondary antibody.

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