Anti-Smad2 antibody [EP784Y]

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Smad2 antibody [EP784Y] (AB40855)

ab40855 at a 1 : 100 dilution staining Smad2 in human prostate carcinoma tissue.

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Smad2 antibody [EP784Y] (AB40855)

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.

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-Smad2 antibody [EP784Y] (AB40855)

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.

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-Smad2 antibody [EP784Y] (AB40855)

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.

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-Smad2 antibody [EP784Y] (AB40855)

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.

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-Smad2 antibody [EP784Y] (AB40855)

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)

• IP

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Immunoprecipitation - Anti-Smad2 antibody [EP784Y] (AB40855)

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.

All lanes:

Immunoprecipitation - Anti-Smad2 antibody [EP784Y] (ab40855)

Predicted band size: 52 kDa

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• WB

Lab

Western blot - Anti-Smad2 antibody [EP784Y] (AB40855)

Diluting and blocking buffer : 5% NFDM /TBST

All lanes:

Western blot - Anti-Smad2 antibody [EP784Y] (ab40855) at 1/2000 dilution

Lane 1:

A-673 (Human muscle Ewing's Sarcoma cell line) whole cell lysate at 20 µg

Lane 2:

HUVEC (Human umbilical vein endothelial cell line) whole cell lysate at 20 µg

Lane 3:

C6 (Rat glial tumor cell line) whole cell lysate at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

Predicted band size: 52 kDa

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• WB

Supplier Data

Western blot - Anti-Smad2 antibody [EP784Y] (AB40855)

The molecular weight observed is consistent with what has been described in the literature (PMID : 34655614).

The up-regulation of pSmad2 is induced by TGF-beta treatment (PMID : 34655614, 24347165).

In Western blot, ab316117 was shown to bind specifically to Smad2. Target of interest was observed at 60 kDa in wild-type Hela cell lysates (lanes 2) with no signal observed at this size in Smad2 knockout cell line lysates (lanes 3-4 KO cell line ab255430/KO cell lysate ab263833 ).

In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control (ab181602) staining at 1/200000 dilution.

In Western blot, Anti-Smad2 antibody [EP784Y] - Total protein control (ab40855) staining at 1/1000 dilution.

All lanes:

Western blot - Anti-Smad2 (phospho S465 + S467) antibody [EPR28610-44] (<a href='/en-us/products/primary-antibodies/smad2-phospho-s465-s467-antibody-epr28610-44-ab316117'>ab316117</a>) at 1/1000 dilution

Lane 1:

Untreated wild-type HeLa (human cervical adenocarcinoma epithelial cell) whole cell lysate (untreated membrane) at 20 µg

Lane 2:

Wild-type Hela treated with 20 ng/ml TGF beta 1 for 60 minutes, whole cell lysate (untreated membrane) at 20 µg

Lane 3:

Untreated Smad2 knockout Hela whole cell lysate (untreated membrane) at 20 µg

Lane 4:

Smad2 knockout Hela treated with 20 ng/ml TGF beta 1 for 60 minutes, whole cell lysate (untreated membrane) at 20 µg

Lane 5:

Untreated wild-type HeLa (human cervical adenocarcinoma epithelial cell) whole cell lysate (alkaline phosphatase treated membrane) at 20 µg

Lane 6:

Wild-type Hela treated with 20 ng/ml TGF beta 1 for 60 minutes, whole cell lysate (alkaline phosphatase treated membrane) at 20 µg

Lane 7:

Untreated Smad2 knockout Hela whole cell lysate (alkaline phosphatase treated membrane) at 20 µg

Lane 8:

Smad2 knockout Hela treated with 20 ng/ml TGF beta 1 for 60 minutes, whole cell lysate (alkaline phosphatase treated membrane) at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/100000 dilution

Observed band size: 60 kDa,36 kDa

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Exposure time: 37s

• WB

Supplier Data

Western blot - Anti-Smad2 antibody [EP784Y] (AB40855)

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.

All lanes:

Western blot - Anti-Smad2 antibody [EP784Y] (ab40855) at 1/2000 dilution

Lane 1:

A549 cell lysate at 20 µg

Lane 2:

Jurkat cell lysate at 20 µg

Lane 2:

Western blot - Human SMAD2 knockout HeLa cell line (<a href='/en-us/products/cell-lines/human-smad2-knockout-hela-cell-line-ab255430'>ab255430</a>)

Lane 3:

Wild-type HeLa cell lysate at 20 µg

Lane 4:

SMAD2 knockout HeLa cell lysate at 20 µg

Secondary

All lanes:

Western blot - Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-irdye-800cw-preadsorbed-ab216773'>ab216773</a>) at 1/20000 dilution

Predicted band size: 52 kDa

Observed band size: 37 kDa,55 kDa

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• WB

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Western blot - Anti-Smad2 antibody [EP784Y] (AB40855)

All lanes:

Western blot - Anti-Smad2 antibody [EP784Y] (ab40855) at 1/500000 dilution

All lanes:

Jurkat cell lysate

Predicted band size: 52 kDa

Observed band size: 58 kDa

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• WB

Lab

Western blot - Anti-Smad2 antibody [EP784Y] (AB40855)

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.

All lanes:

Western blot - Anti-Smad2 antibody [EP784Y] (ab40855) at 1/1000 dilution

Lane 1:

Wild-type HeLa whole cell lysate at 20 µg

Lane 2:

Smad2 knockout HeLa whole cell lysate at 20 µg

Lane 3:

A549 whole cell lysate at 20 µg

Predicted band size: 52 kDa

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• WB

Lab

Western blot - Anti-Smad2 antibody [EP784Y] (AB40855)

Blocking and diluting buffer : 5% NFDM/TBST

All lanes:

Western blot - Anti-Smad2 antibody [EP784Y] (ab40855) at 1/10000 dilution

Lane 1:

Jurkat (Human T cell leukemia cell line from peripheral blood) whole cell lysate at 20 µg

Lanes 2 - 3:

HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 20000 µg

Predicted band size: 52 kDa

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• ChIC/CUT&RUN-seq

Lab

ChIC/CUT&RUN sequencing - Anti-Smad2 antibody [EP784Y] (AB40855)

CUT&RUN profiling with Smad2 antibody reveals the expected genomic enrichment pattern in cells. Representative genome browser tracks show CUT&RUN data generated using the CUTANA™ CUT&RUN Kit (EpiCypher 14-1048) with Smad2 antibody (Abcam ab40855, 0.5 µg). 500,000 HeLa cells were used per reaction. IgG, H3K4me3, and H3K27me3 antibodies were included as controls to assess non-specific background, active promoters, and repressed chromatin, respectively. Libraries were prepared using the CUTANA™ CUT&RUN Library Prep Kit (EpiCypher 14-1001). Sequencing was performed with paired-end 50 bp reads, and data were processed on CUTANA™ Cloud (cloud.epicypher.com) by alignment to the hg38 genome. Images were generated using Integrative Genomics Viewer (IGV, Broad Institute).

• ChIC/CUT&RUN-seq

Lab

ChIC/CUT&RUN sequencing - Anti-Smad2 antibody [EP784Y] (AB40855)

ChIC/CUT&RUN was performed using a pAG-MNAse at a final concentration of 700 ng/mL, 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 found in the Protocol booklet in the Product Protocol section. The University of Geneva owns patents relevant to ChIC (Chromatin Immuno-Cleavage) methods.

• ChIC/CUT&RUN-seq

Lab

ChIC/CUT&RUN sequencing - Anti-Smad2 antibody [EP784Y] (AB40855)

CUT&RUN profiling with Smad2 antibody demonstrates robust genome-wide enrichment in cells. Heatmaps of genome-wide signal flanking annotated transcription start sites (TSSs, +/- 2 kbp) display CUT&RUN data generated using the CUTANA™ CUT&RUN Kit (EpiCypher 14-1048) with Smad2 antibody (Abcam ab40855, 0.5 µg). 500,000 HeLa cells were used per reaction. IgG antibody was included as a negative control to assess non-specific background. Libraries were prepared using the CUTANA™ CUT&RUN Library Prep Kit (EpiCypher 14-1001). Sequencing was performed with paired-end 50 bp reads, and data were processed on CUTANA™ Cloud (cloud.epicypher.com) by alignment to the hg38 genome. Heatmaps were generated using deepTools (Ramírez et al., Nucleic Acids Res. 2014; PMID : 24799436). Row-linked data are ranked by intensity relative to Smad2, with red indicating high localized enrichment and blue denoting background.