Anti-Smad2 antibody [EP567Y] - BSA and Azide free

• ICC/IF

Unknown

Immunocytochemistry/ Immunofluorescence - Anti-Smad2 antibody [EP567Y] - BSA and Azide free (AB216454)

Immunofluorescent staining of A673 cells, fixed with 4% PFA, using purified ab33875 at a dilution of 1/300. An Alexa Fluor^® 555 goat anti-rabbit was used at 1/200. The negative control is shown in the bottom right hand panel - for the negative control, purified ab33875 was used at a dilution of 1/200 followed by an Alexa Fluor^® 555 goat anti-mouse antibody at a dilution of 1/500.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab33875).

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-Smad2 antibody [EP567Y] - BSA and Azide free (AB216454)

Overlay histogram showing Jurkat 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 the isotype control (green line).

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab33875).

• Flow Cyt (Intra)

Unknown

Flow Cytometry (Intracellular) - Anti-Smad2 antibody [EP567Y] - BSA and Azide free (AB216454)

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.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab33875).

• WB

Lab

Western blot - Anti-Smad2 antibody [EP567Y] - BSA and Azide free (AB216454)

This data was developed using the same antibody clone in a different buffer formulation (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] (<a href='/en-us/products/primary-antibodies/smad2-antibody-ep567y-ab33875'>ab33875</a>) 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

false

• ChIC/CUT&RUN-seq

Lab

ChIC/CUT&RUN sequencing - Anti-Smad2 antibody [EP567Y] - BSA and Azide free (AB216454)

This data was developed using the same antibody clone in a different buffer formulation (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.