Anti-Smad3 antibody [EP568Y]

• Flow Cyt (Intra)

Unknown

Flow Cytometry (Intracellular) - Anti-Smad3 antibody [EP568Y] (AB40854)

Intracellular Flow Cytometry analysis of HT-29 cells labelling Smad3 with purified ab40854 at 1/210 (red). Cells were fixed with 2% paraformaldehyde. A FITC-conjugated goat anti-rabbit IgG (1/150) was used as the secondary antibody. Green - Isotype control, rabbit monoclonal IgG.

• Flow Cyt (Intra)

Unknown

Flow Cytometry (Intracellular) - Anti-Smad3 antibody [EP568Y] (AB40854)

Overlay histogram showing HCT116 cells stained with unpurifiedab40854 (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 (ab40854, 1/100 dilution) for 30 min at 22°C. The secondary antibody used was DyLight^® 488 goat anti-rabbit IgG (H+L) (ab96899) at 1/500 dilution for 30 min at 22°C. Isotype control antibody (black line) was rabbit IgG (monoclonal) (1μg/1x10⁶ cells) used under the same conditions. Acquisition of >5,000 events was performed.

• ICC/IF

Unknown

Immunocytochemistry/ Immunofluorescence - Anti-Smad3 antibody [EP568Y] (AB40854)

Immunocytochemsitry/Immunofluorescence analysis of HepG2 cells labelling Smad3 (green) with purified ab40854 at 1/2000 . Cells were fixed with 4% paraformaldehyde. An Alexa Fluor^® 488-conjugated goat anti-rabbit IgG (1/200) was used as the secondary antibody (green, left panel). Counterstained with DAPI (blue, right panel).

• ICC/IF

AbReview43264****

Immunocytochemistry/ Immunofluorescence - Anti-Smad3 antibody [EP568Y] (AB40854)

ab40854 staining Smad3 in human granulosa cells by ICC/IF (Immunocytochemistry/immunofluorescence). Cells were fixed with paraformaldehyde, permeabilized with ethanol and triton and blocked for 1 hour at 26°C. Samples were incubated with primary antibody (1/200) for 16 hours at 4°C. An undiluted IRDye^® 800CW-conjugated goat anti-rabbit IgG (H+L) polyclonal was used as the secondary antibody. Left - negative control (4 replicates).

This image is courtesy of an Abreview submitted by Francesco Elia Marino

• WB

Lab

Western blot - Anti-Smad3 antibody [EP568Y] (AB40854)

False colour image of Western blot : Anti-Smad3 antibody [EP568Y] staining at 1/1000 dilution, shown in green; Mouse anti-GAPDH antibody [6C5] (ab8245) loading control staining at 1/20000 dilution, shown in red. In Western blot, ab40854 was shown to bind specifically to Smad3. A band was observed at 50 kDa in wild-type A549 cell lysates with no signal observed at this size in SMAD3 CRISPR-Cas9 edited cell line ab277888 (CRISPR-Cas9 edited cell lysate None). The band observed in the CRISPR-Cas9 edited lysate lane below 50 kDa is likely to represent a truncated form of Smad3. This has not been investigated further and the functional properties of the gene product have not been determined. To generate this image, wild-type and SMAD3 CRISPR-Cas9 edited A549 cell lysates were analysed. First, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 3 % milk in TBS-0.1 % Tween^® 20 (TBS-T) before incubation with primary antibodies overnight at 4 °C. Blots were washed four times in TBS-T, incubated with secondary antibodies for 1 h at room temperature, washed again four times then imaged. Secondary antibodies used were Goat anti-Rabbit IgG H&L 800CW and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution.

All lanes:

Western blot - Anti-Smad3 antibody [EP568Y] (ab40854) at 1/1000 dilution

Lane 1:

Wild-type A549 cell lysate at 20 µg

Lane 2:

Western blot - Human SMAD3 knockout A549 cell line (<a href='/en-us/products/cell-lines/human-smad3-knockout-a549-cell-line-ab277888'>ab277888</a>)

Lane 2:

SMAD3 CRISPR-Cas9 edited A549 cell lysate at 20 µg

Secondary

Lanes 1 - 2:

Goat anti-Rabbit IgG H&L 800CW at 1/20000 dilution

Lanes 1 - 2:

Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution

Predicted band size: 48 kDa

Observed band size: 50 kDa

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• WB

Lab

Western blot - Anti-Smad3 antibody [EP568Y] (AB40854)

Lanes 1 - 4 : Merged signal (red and green). Green - ab40854 observed at 48 kDa. Red - loading control, ab8245 (Mouse anti-GAPDH antibody [6C5]) observed at 37kDa.

ab40854 was shown to react with Smad3 in western blot. Membranes were blocked in 3% milk in TBS-T (0.1% Tween^®) before incubation with ab40854 and ab8245 (Mouse anti-GAPDH antibody [6C5]) overnight at 4°C at a 1 in 1000 Dilution and a 1 in 20000 dilution respectively. Blots were incubated 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 in 20000 dilution for 1 hour at room temperature before imaging.

All lanes:

Western blot - Anti-Smad3 antibody [EP568Y] (ab40854) at 1/1000 dilution

Lane 1:

Wild-type A549 (Human lung carcinoma cell line) whole cell lysate at 20 µg

Lane 2:

Western blot - Human SMAD3 knockout A549 cell lysate (<a href='/en-us/products/cell-lysates/human-smad3-knockout-a549-cell-lysate-ab264513'>ab264513</a>)

Lane 3:

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

Lane 4:

Human Kidney cell lysate at 20 µg

Predicted band size: 48 kDa

Observed band size: 50 kDa

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• WB

Lab

Western blot - Anti-Smad3 antibody [EP568Y] (AB40854)

Blocking buffer and concentration : 5% NFDM/TBST.

Diluting buffer and concentration : 5% NFDM /TBST.

All lanes:

Western blot - Anti-Smad3 antibody [EP568Y] (ab40854) at 1/5300 dilution

Lane 1:

HT-29 cell lysate at 10 µg

Lane 2:

HT-1080 cell lysate at 10 µg

Secondary

All lanes:

Peroxidase-conjugated goat anti-rabbit IgG (H+L) at 1/1000 dilution

Predicted band size: 48 kDa

Observed band size: 58 kDa

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• WB

Lab

Western blot - Anti-Smad3 antibody [EP568Y] (AB40854)

This blot was produced using 4-20% SDS-PAGE containing 15 μg of HT-29 whole cell lysate per lane at 150V for 1hr before being transferred onto a 0.45 μm PVDF membrane at 75V for 1hr. The membrane was then blocked for 1hr using 5% NFDM/TBST, then incubated with ab40854 (1/5000) at room temperature for 1hr. After being washed three times in TBST, the membrane was incubated with Peroxidase conjugated goat anti-rabbit IgG (H+L) (ab97051) at 1/20,000 dilution for 1hr at room temperature. The membrane was washed three times again. Then the signal was developed using the ECL technique. ab40854 was stored at a range of temperatures (+4°C, +22°C, +37°C) for 1 week before being tested in WB. The image shows the band intensity remains relatively constant across all storage temperatures, demonstrating that antibody activity is not affected.

All lanes:

Western blot - Anti-Smad3 antibody [EP568Y] (ab40854) at 1/5000 dilution

All lanes:

HT-29 whole cell lysate at 15 µg with NDFM/TBST

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

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Exposure time: 10s

• ChIC/CUT&RUN-seq

Lab

ChIC/CUT&RUN sequencing - Anti-Smad3 antibody [EP568Y] (AB40854)

ChIC/CUT&RUN was performed using a pAG-MNase at a final concentration of 700 ng/mL, 2.5 x 10^5 A549 (Human lung carcinoma cell line) cells treated with hTGF-β1 (7 ng/mL 1 h) and 5 µg of ab40854 [EP568Y]. 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. The University of Geneva owns patents relevant to ChIC (Chromatin Immuno-Cleavage) methods.

• ChIC/CUT&RUN-seq

Lab

ChIC/CUT&RUN sequencing - Anti-Smad3 antibody [EP568Y] (AB40854)

ChIC/CUT&RUN was performed using a pAG-MNase at a final concentration of 700ng/mL, 2.5 x 10^5 A549 (Human lung carcinoma cell line) cells treated with hTGF-β1 (7 ng/mL 1 h) and 5 µg of ab40854 [EP568Y]. 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. The University of Geneva owns patents relevant to ChIC (Chromatin Immuno-Cleavage) methods.

• ChIC/CUT&RUN-seq

Supplier Data

ChIC/CUT&RUN sequencing - Anti-Smad3 antibody [EP568Y] (AB40854)

CUT&RUN profiling with Smad3 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 Smad3 antibody (Abcam ab40854, 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

Supplier Data

ChIC/CUT&RUN sequencing - Anti-Smad3 antibody [EP568Y] (AB40854)

CUT&RUN profiling with Smad3 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 Smad3 antibody (Abcam ab40854, 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 ChAsE (Younesy et al., Bioinformatics 2016; PMID 27378294). Row-linked data are ranked by intensity relative to Smad3, with red indicating high localized enrichment and blue denoting background.

• ChIC/CUT&RUN-seq

Lab

ChIC/CUT&RUN sequencing - Anti-Smad3 antibody [EP568Y] (AB40854)

ChIC/CUT&RUN was performed using a pAG-MNase at a final concentration of 700 ng/mL, 2.5 x 10^5 A549 (Human lung carcinoma cell line) cells treated with hTGF-β1 (7 ng/mL 1 h) and 5 µg of ab40854 [EP568Y]. 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. The University of Geneva owns patents relevant to ChIC (Chromatin Immuno-Cleavage) methods.

• WB

Lab

Western blot - Anti-Smad3 antibody [EP568Y] (AB40854)

Blocking buffer and concentration : 5% NFDM/TBST.

Diluting buffer and concentration : 5% NFDM /TBST.

All lanes:

Western blot - Anti-Smad3 antibody [EP568Y] (ab40854) at 1/5300 dilution

All lanes:

Rat liver tissue lysate at 10 µg

Secondary

All lanes:

Peroxidase-conjugated goat anti-rabbit IgG (H+L) at 1/1000 dilution

Predicted band size: 48 kDa

Observed band size: 58 kDa

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• sELISA

Unknown

Sandwich ELISA - Anti-Smad3 antibody [EP568Y] (AB40854)

Standard Curve for Smad3 (Analyte : Smad3 protein (His tag) (ab89353, unpurified)); dilution range 1pg/ml to 1μg/ml using Capture Antibody Mouse monoclonal [AF9F7] to Smad3 (ab75512) at 5μg/ml and Detector Antibody Rabbit monoclonal [EP568Y] to Smad3 (ab40854) at 0.5μg/ml.

• WB

Unknown

Western blot - Anti-Smad3 antibody [EP568Y] (AB40854)

All lanes:

Western blot - Anti-Smad3 antibody [EP568Y] (ab40854) at 1/5000 dilution

All lanes:

Jurkat cell lysate at 10 µg

Predicted band size: 48 kDa

Observed band size: 55 kDa

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