Chromatin was prepared from SK-OV-3 (Human ovarian cancer cell line) cells according to the Abcam X-ChIP protocol. Cells were fixed with formaldehyde for 10 minutes. The ChIP was performed with 25µg of chromatin, 5µg of ab32058 (red, and 20µl of Anti rabbit IgG sepharose beads. 5μg of rabbit normal IgG was added to the beads control (grey).  The immunoprecipitated DNA was quantified by real time PCR (Sybr green approach).

ChIP was performed according to the literature (PMID: 23770046).

Lane 1: Wild-type HAP1 whole cell lysate (20 µg)
Lane 2: Empty
Lane 3: Sumo 1 knockout HAP1 whole cell lysate (20 µg)
Lane 4: Empty

Lanes 1 - 4: Merged signal (red and green). Green - ab32058 observed at 16 kDa. Red - loading control, ab8245, observed at 37 kDa.

ab32058 was shown to react with Sumo 1 in wild-type HAP1 cells along with additional cross-reactive bands. No band was observed when Sumo 1 knockout samples were used. Wild-type and Sumo 1 knockout samples were subjected to SDS-PAGE. Samples were incubated with ab32058 and ab8245 (Mouse anti GAPDH loading control) overnight at 4°C at a 1/1000 dilution and 1/10000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preabsorbed ab216773 and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed ab216776 secondary antibodies at 1/10000 dilution for 1 hour at room temperature before imaging.

HSV-1 MA pattern corresponds to vDCP-NBs and contains SUMO proteins

Immuno-DNA-FISH showing HSV-1 genomes (red), small ubiquitin modifier (SUMO) proteins (green), and cellular chromatin (DAPI, blue/grey).  SUMO-1 in (i) non-infected neurons, (ii) RC-containing neurons and (iii, iv) MA/vDCP-NBs or S/vDCP-NB-containing neurons.

(From Figure 2C of Maroui et al)

ab32058 staining Sumo 1 in HeLa (Human epithelial cell line from cervix adenocarcinoma) cells by ICC/IF (Immunocytochemistry/immunofluorescence). Cells were fixed with 4% Paraformaldehyde and permeabilized with 0.1% Triton X-100. Samples were incubated with primary antibody at a dilution of 1/500. A goat anti rabbit IgG (Alexa Fluor® 488) (ab150077) was used as the secondary antibody at a dilution of 1/1000. ab195889 was used as a counterstain for primary antibody ab133645 at 1/200. DAPI was used as a nuclear counterstain and PBS as a negative control.

ab32058 staining Sumo 1 in mouse kidney tissue sections by Immunohistochemistry (IHC-P - paraformaldehyde-fixed, paraffin-embedded sections). Tissue was fixed with paraformaldehyde and antigen retrieval was by heat mediation in a EDTA buffer. Samples were incubated with primary antibody at a dilution of 1/250. A goat anti-rabbit IgG H&L (HRP) ab97051 was used as the secondary antibody at a dilution of 1/500.

Negative control 1: PBS in place of primary antibody.

ab32058 staining Sumo 1 in HeLa (Human epithelial cell line from cervix adenocarcinoma) cells by flow cytometry. Cells were fixed with 4% paraformaldehyde and the sample was incubated with the primary antibody at a dilution of 1/20. A goat anti rabbit IgG (Alexa Fluor® 488) at a dilution of 1/2000 was used as the secondary antibody.

Isoytype control: Rabbit monoclonal IgG (Black)

Unlabelled control: Cell without incubation with primary antibody and secondary antibody (Blue)

ab32058 immunoprecipitating Sumo 1. 10µg of NIH/3T3 (Mouse embryonic fibroblast) cell lysate was incubated with primary antibody at a dilution of 1/20 and VeriBlot for IP Detection Reagent (HRP) (ab131366) at a dilution of 1/1000.

Lane 1: NIH/3T3 whole cell lysate 10ug
Lane 2: ab32058 IP in NIH/3T3 whole cell lysate
Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab32058 in NIH/3T3 (Mouse embryonic fibroblast) whole cell lysate

ab32058 staining Sumo 1 in rat stomach tissue sections by Immunohistochemistry (IHC-P - paraformaldehyde-fixed, paraffin-embedded sections). Tissue was fixed with paraformaldehyde and antigen retrieval was by heat mediation in a EDTA buffer. Samples were incubated with primary antibody at a dilution of 1/250. A goat anti-rabbit IgG H&L (HRP) ab97051 was used as the secondary antibody at a dilution of 1/500.

Negative control 1: PBS in place of primary antibody.

ab32058 staining Sumo 1 in human bladder carcinoma tissue sections by Immunohistochemistry (IHC-P - paraformaldehyde-fixed, paraffin-embedded sections). Tissue was fixed with paraformaldehyde and antigen retrieval was by heat mediation in a EDTA buffer. Samples were incubated with primary antibody at a dilution of 1/250. A goat anti-rabbit IgG H&L (HRP) ab97051 was used as the secondary antibody at a dilution of 1/500.

Negative control 1: PBS in place of primary antibody.

ab32058 staining Sumo 1 in human endometrium tissue sections by Immunohistochemistry (IHC-P - paraformaldehyde-fixed, paraffin-embedded sections). Tissue was fixed with paraformaldehyde and antigen retrieval was by heat mediation in a EDTA buffer. Samples were incubated with primary antibody at a dilution of 1/250. A goat anti-rabbit IgG H&L (HRP) ab97051 was used as the secondary antibody at a dilution of 1/500.

Negative control 1: PBS in place of primary antibody.

All lanes : Anti-Sumo 1 antibody [Y299] - ChIP Grade (ab32058) at 1/5000 dilution

Lane 1 : HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysate
Lane 2 : A549 (Human lung carcinoma epithelial cell) whole cell lysate
Lane 3 : NIH/3T3 (Mouse embryonic fibroblast) whole cell lysate
Lane 4 : C6 (Rat glial tumor glial cell) whole cell lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution

Predicted band size: 12 kDa

Blocking and diluting buffer: 5% NFDM/TBST

Immunofluorescence analysis of ICP0-null mutant HSV-1 infected HepaRG cells, staining Sumo1 (green) with ab32058. An AlexaFluor®-conjugated goat anti-rabbit IgG was used as the seconday antibody.

All lanes : Anti-Sumo 1 antibody [Y299] - ChIP Grade (ab32058) at 1/1000 dilution

Lane 1 : HeLa (Human epithelial cell line from cervix adenocarcinoma) cell lysate
Lane 2 : NIH/3T3 (Mouse embryo fibroblast cell line) cell lysate

Predicted band size: 12 kDa
Observed band size: 12,80 kDa why is the actual band size different from the predicted?

Flow Cytometry analysis of HeLa (human cervix adenocarcinoma) cells labelling Sumo 1 with ab32058 at 1/20 (red). Cells were fixed with 4% paraformaldehyde and permeabilised with 90% methanol. An Alexa Fluor^® 488-conjugated goat anti-rabbit IgG (1/2000) was used as the secondary antibody. Black - Isotype control, rabbit monoclonal IgG. Blue - Unlabelled control, cells without incubation with primary and secondary antibodies.

IHC of paraffin-embedded human lung carcinoma using anti-SUMO 1 (ab32058) diluted 1:250

Immunofluorescent staining of HeLa (Human epithelial cell line from cervix adenocarcinoma) cells using anti-SUMO 1 (ab32058) diluted 1/250.