developed using the ECL techniquePerformed under reducing conditions.

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.

Image courtesy of Human Protein Atlasab3742 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

IHC image of Sumo 2+3 staining in human colon adenocarcinoma 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 ab3742, 1µg/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.