Anti-Smad3 antibody [EPR19686] - ChIP Grade

• IP

Supplier Data

Immunoprecipitation - Anti-Smad3 antibody [EPR19686] - ChIP Grade (AB208182)

Smad3 was immunoprecipitated from 0.35 mg of HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate with ab208182 at 1/30 dilution.

Western blot was performed from the immunoprecipitate using ab208182 at 1/1000 dilution.

VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/10000 dilution.

Lane 1 : HeLa whole cell lysate 10μg (Input).

Lane 2 : ab208182 IP in HeLa whole cell lysate.

Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab208182 in HeLa whole cell lysate.

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

Exposure time : 3 minutes.

All lanes:

Immunoprecipitation - Anti-Smad3 antibody [EPR19686] - ChIP Grade (ab208182)

Predicted band size: 48 kDa

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• ChIP

Unknown

ChIP - Anti-Smad3 antibody [EPR19686] - ChIP Grade (AB208182)

Chromatin was prepared from HaCaT (Human keratinocyte cell line) cells treated with 7ng/ml TGF-β for 1h and non-treated according to the Abcam X-ChIP protocol. Cells were fixed with formaldehyde for 10 minutes. The ChIP was performed with 25μg of chromatin, 2μg of ab208182 (red), and 20μl of protein A/G beads. 2μg of rabbit normal IgG was added to the beads control (grey). The immunoprecipitated DNA was quantified by real time PCR (Sybr green approach).

The ChIP condition performed here is similar to the literature (PMID : 18245174).

• WB

Lab

Western blot - Anti-Smad3 antibody [EPR19686] - ChIP Grade (AB208182)

Lanes 1 - 4 : Merged signal (red and green). Green - ab208182 observed at 50 kDa. Red - loading control, ab8245 (Mouse anti-GAPDH antibody [6C5]) observed at 37kDa.

ab208182 was shown to react with Smad3 in western blot. Membranes were blocked in 3% milk in TBS-T (0.1% Tween^®) before incubation with ab208182 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 [EPR19686] - ChIP Grade (ab208182) 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 [EPR19686] - ChIP Grade (AB208182)

Lanes 1 - 4 : Merged signal (red and green). Green - ab208182 observed at 55 kDa. Red - loading control, ab8245 observed at 37 kDa.

ab208182 was shown to react with Smad3 in wild-type HeLa cells. Loss of signal was observed when knockout cell line ab255431 (knockout cell lysate ab263834) was used. Wild-type and Smad3 knockout samples were subjected to SDS-PAGE. ab208182 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) were incubated overnight at 4°C at 1 in 1000 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-Smad3 antibody [EPR19686] - ChIP Grade (ab208182) at 1/1000 dilution

Lane 1:

Wild-type A549 cell lysate at 20 µg

Lane 2:

SMAD3 knockout A549 cell lysate at 20 µg

Lane 3:

Wild-type HeLa cell lysate at 20 µg

Lane 4:

Western blot - Human SMAD3 knockout HeLa cell lysate (<a href='/en-us/products/cell-lysates/human-smad3-knockout-hela-cell-lysate-ab263834'>ab263834</a>)

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: 48 kDa

Observed band size: 37 kDa,74 kDa

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• WB

Supplier Data

Western blot - Anti-Smad3 antibody [EPR19686] - ChIP Grade (AB208182)

Blocking/Dilution buffer : 5% NFDM/TBST.

Exposure time : Lane 1/6 : 3 minutes; Lane 2-5 : 15 seconds.

All lanes:

Western blot - Anti-Smad3 antibody [EPR19686] - ChIP Grade (ab208182) at 1/1000 dilution

Lane 1:

HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate at 10 µg

Lane 2:

Jurkat (Human T cell leukemia cell line from peripheral blood) whole cell lysate at 10 µg

Lane 3:

HEK-293 (Human epithelial cell line from embryonic kidney) whole cell lysate at 10 µg

Lane 4:

K562 (Human chronic myelogenous leukemia cell line from bone marrow) whole cell lysate at 10 µg

Lane 5:

BxPC-3 (Human pancreas adenocarcinoma cell line) whole cell lysate at 10 µg

Lane 6:

Human fetal kidney lysate at 10 µg

Secondary

Lanes 1 - 5:

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

Lane 6:

Goat Anti-Rabbit IgG Peroxidase Conjugate, specific to the non-reduced form of IgG at 1/10000 dilution

Predicted band size: 13 kDa,48 kDa

Observed band size: 55 kDa

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• WB

Supplier Data

Western blot - Anti-Smad3 antibody [EPR19686] - ChIP Grade (AB208182)

Blocking/Dilution buffer : 5% NFDM/TBST.

Human Smad3 recombinant protein contains aa2-227 with GST and His-Tag®. This protein was made-in house.

All lanes:

Western blot - Anti-Smad3 antibody [EPR19686] - ChIP Grade (ab208182) at 1/1000 dilution

Lane 1:

Human Smad3 recombinant protein at 0.01 µg

Lane 2:

Human Smad2 active protein at 0.01 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

Predicted band size: 48 kDa

Observed band size: 74 kDa

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Exposure time: 1s

• WB

Lab

Western blot - Anti-Smad3 antibody [EPR19686] - ChIP Grade (AB208182)

False colour image of Western blot : Anti-Smad3 antibody [EPR19686] - ChIP Grade 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, ab208182 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 [EPR19686] - ChIP Grade (ab208182) at 1/1000 dilution

Lane 1:

Wild-type A549 cell lysate at 20 µg

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

Observed band size: 50 kDa

false

• WB

Supplier Data

Western blot - Anti-Smad3 antibody [EPR19686] - ChIP Grade (AB208182)

Blocking/Dilution buffer : 5% NFDM/TBST.

Exposure time : Lane 1/2 : 30 seconds; Lane 3-6 : 3 minutes.

All lanes:

Western blot - Anti-Smad3 antibody [EPR19686] - ChIP Grade (ab208182) at 1/1000 dilution

Lane 1:

C6 (Rat glial tumor cell line) whole cell lysate at 10 µg

Lane 2:

PC-12 (Rat adrenal gland pheochromocytoma cell line) whole cell lysate at 10 µg

Lane 3:

NIH/3T3 (Mouse embryonic fibroblast cell line) whole cell lysate at 10 µg

Lane 4:

Rat spleen lysate at 10 µg

Lane 5:

Rat kidney lysate at 10 µg

Lane 6:

Mouse spleen lysate at 10 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

Predicted band size: 48 kDa

Observed band size: 55 kDa

false