Anti-Smad2 antibody [EP567Y]

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-Smad2 antibody [EP567Y] (AB33875)

Immunocytochemistry/Immunofluorescent analysis of HeLa (human cervix adenocarcinoma epithelial) cells labeling Smad2 with ab33875 at a dilution of 1/500. ab150077, an Alexa Fluor^® 488 goat anti-rabbit was used at 1/1000 was used as the secondary antibody. Cells were fixed with 4% Paraformaldehyde and permeabilised with 0.1% tritonX-100. Counterstain antibody : ab195889, anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594) at 1/200.

Secondary antibody only negative control is shown in the bottom panels.

Confocal image showing mainly nuclear staining on HeLa cells after the treatment with TGF-b (10ng/mL) for 1 hour.

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-Smad2 antibody [EP567Y] (AB33875)

Overlay histogram showingJurkat cells stained with purified ab33875 (pink line) at a dilution of 1/110. The cells were fixed with 2% PFA.FITC goat anti-rabbit was used at a dilution of 1/150 and rabbit monoclonal IgG was used as theisotype control (green line).

• Flow Cyt (Intra)

Unknown

Flow Cytometry (Intracellular) - Anti-Smad2 antibody [EP567Y] (AB33875)

Overlay histogram showing PC3 cells stained with unpurified ab33875 (red line). The cells were fixed with 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 (ab33875, 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 monoclonal IgG (1μg/1x10⁶ cells) used under the same conditions. Acquisition of >5,000 events was performed. This antibody gave a decreased signal in PC3 cells fixed with 4% paraformaldehyde (10 min)/permeabilized in 0.1% PBS-Tween used under the same conditions.

• WB

Lab

Western blot - Anti-Smad2 antibody [EP567Y] (AB33875)

Lanes 1 - 3 : Merged signal (red and green). Green - ab33875 observed at 52 kDa. Red - loading control, ab9484, observed at 37 kDa.

ab33875 was shown to specifically react with Smad2 in wild-type WT HeLa cells as signal was lost in SMAD2 knockout cells. Wild-type and SMAD2 knockout samples were subjected to SDS-PAGE. ab33875 and ab9484 (Mouse anti-GAPDH loading control) were incubated overnight at 4°C at 1/1000 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 [EP567Y] (ab33875) 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

Observed band size: 52 kDa

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• WB

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Western blot - Anti-Smad2 antibody [EP567Y] (AB33875)

Blocking buffer : 5% NFDM/TBST

Dilution buffer : 5% NFDM/TBST

All lanes:

Western blot - Anti-Smad2 antibody [EP567Y] (ab33875) at 1/2000 dilution

All lanes:

Jurkat cell lysate at 10 µg

Secondary

All lanes:

HRP goat anti-rabbit (H+L) at 1/1000 dilution

Predicted band size: 52 kDa

Observed band size: 58 kDa

false

• WB

Lab

Western blot - Anti-Smad2 antibody [EP567Y] (AB33875)

Blocking buffer : 5% NFDM/TBST

Dilution buffer : 5% NFDM/TBST

All lanes:

Western blot - Anti-Smad2 antibody [EP567Y] (ab33875) at 1/1000 dilution

All lanes:

Jurkat cell lysate at 10 µg

Secondary

All lanes:

HRP goat anti-rabbit (H+L) at 1/1000 dilution

Predicted band size: 52 kDa

Observed band size: 58 kDa

false

• WB

Lab

Western blot - Anti-Smad2 antibody [EP567Y] (AB33875)

Lanes 1 - 4 : Merged signal (red and green). Green - ab33875 observed at 58 kDa. Red - loading control, ab8245 observed at 37 kDa.

ab33875 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. ab33875 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-Smad2 antibody [EP567Y] (ab33875) at 1/1000 dilution

Lane 1:

Wild-type 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,58 kDa

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• ChIC/CUT&RUN-seq

Lab

ChIC/CUT&RUN sequencing - Anti-Smad2 antibody [EP567Y] (AB33875)

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 ab33875 [EP567Y]. 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.

• WB

Lab

Western blot - Anti-Smad2 antibody [EP567Y] (AB33875)

Blocking buffer : 5% NFDM/TBST

Dilution buffer : 5% NFDM/TBST

All lanes:

Western blot - Anti-Smad2 antibody [EP567Y] (ab33875) at 1/1000 dilution

All lanes:

RAW264.7 at 10 µg

Secondary

All lanes:

HRP goat anti-rabbit (H+L) at 1/1000 dilution

Predicted band size: 52 kDa

Observed band size: 58 kDa

false

• WB

Lab

Western blot - Anti-Smad2 antibody [EP567Y] (AB33875)

Blocking buffer : 5% NFDM/TBST

Dilution buffer : 5% NFDM/TBST

All lanes:

Western blot - Anti-Smad2 antibody [EP567Y] (ab33875) at 1/500 dilution

All lanes:

RAW264.7 cell lysate at 10 µg

Secondary

All lanes:

HRP goat anti-rabbit (H+L) at 1/1000 dilution

Predicted band size: 52 kDa

Observed band size: 58 kDa

false