Recombinant Anti-Smad2 antibody [EP784Y] (ab40855)

ChIC/CUT&RUN was performed using a pAG-MNAse at a final concentration of 700 ng/µL, 2.5 x 10^5 HaCaT (Human keratinocyte cell line) cells (treated with 7ng/ml TGF-β for 1h) and 5 µg of ab40855 [EP784Y]. The resulting DNA was sequenced on the Illumina NovaSeq 6000 to a depth of 10 million reads. The negative IgG control ab172730 is also shown.
Additional screenshots of mapped reads can be downloaded here. The University of Geneva owns patents relevant to ChIC (Chromatin Immuno-Cleavage) methods.

Lanes 1 - 4: Merged signal (red and green). Green - ab40855 observed at 58 kDa. Red - loading control, ab8245 observed at 37 kDa.  

 ab40855 was shown to react with Smad2 in wild-type HeLa. Loss of signal was observed when knockout cell line ab255430 (knockout cell lysate ab263833) was used. Wild-type and Smad2 knockout samples were subjected to SDS-PAGE. ab40855 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) were incubated overnight at 4°C at 1 in 2000 dilution and 1 in 20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preadsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preadsorbed (ab216776) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.

Lanes 1 - 3: Merged signal (red and green). Green - ab40855 observed at 58 kDa. Red - loading control, ab9484, observed at 37 kDa.

ab40855 was shown to specifically react with Smad2 in wild-type HeLa cells as signal was lost in Smad2 knockout cells. Wild-type and SMAD2 knockout samples were subjected to SDS-PAGE. Ab40855 and ab9484 (Mouse anti-GAPDH loading control) were incubated overnight at 4°C at 1/10000 dilution and 1/20000 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 1/20000 dilution for 1 hour at room temperature before imaging.

Diluting and blocking buffer: 5% NFDM /TBST

ab40855 staining Smad2 in HeLa (human 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 1/100. 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 ab40855 at 1/1000. DAPI was used as a nuclear counterstain and PBS as a negative control.

Blocking and diluting buffer: 5% NFDM/TBST

ab40855 stainingSmad2 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 1/50. A ImmunoHistoProbe one step HRP Polymer was used as a secondary antibody, ready to use.

Negative control 1: PBS in place of primary antibody.

ab40855 (purified) at 1/20 immunoprecipitating EGFR in HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate.

Lane 1 (input): HeLa whole cell lysate (10µg)

Lane 2 (+): ab40855 + HeLa whole cell lysate.

Lane 3 (-): Rabbit monoclonal IgG (ab172730) instead of ab40855 in HeLa whole cell lysate.

For western blotting, ab131366 VeriBlot for IP (HRP) was used for detection (1/1000).

Blocking/Diluting buffer and concentration: 5% NFDM/TBST.

ab40855 staining Smad2in the human cell line HeLa (Human epithelial cell line from cervix adenocarcinoma) by intracellular 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) 

Immunofluorescence staining of HeLa cells with purified ab40855 at a working dilution of 1/500, counter-stained with DAPI. The secondary antibody was an Alexa Fluor^® 488 conjugated goat anti-rabbit (ab150077), used at a dilution of 1/1000. The cells were fixed in 4% PFA and permeabilized using 0.1% Triton X 100. The negative control is shown in bottom right hand panel - for the negative control, PBS was used instead of the primary antibody.

Overlay histogram showing PC3 cells stained with ab40855 (red line). The cells were fixed with 80% methanol (5 min) and then permeabilized with 0.1% PBS-Tween for 20 min. The cells were then incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions followed by the antibody (ab40855, 1/100 dilution) for 30 min at 22°C. The secondary antibody used was Alexa Fluor^® 488 goat anti-rabbit IgG (H&L) (ab150077) at 1/2000 dilution for 30 min at 22°C. Isotype control antibody (black line) was rabbit IgG (monoclonal) (1µg/1x10⁶ cells) used under the same conditions. Unlabelled sample (blue line) was also used as a control. Acquisition of >5,000 events were collected using a 20mW Argon ion laser (488nm) and 525/30 bandpass filter.