Anti-Smad2 + Smad3 antibody [EPR19557] - BSA and Azide free

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-Smad2 + Smad3 antibody [EPR19557] - BSA and Azide free (AB236030)

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized HeLa (Human epithelial cell line from cervix adenocarcinoma) cells labeling Smad2 + Smad3 with ab207447 at 1/500 dilution, followed by Goat Anti-Rabbit IgG (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution (green). Confocal image showing signal translocation from cytoplasm to nucleus after TGF-beta (10ng/ml, 1h) treatment in HeLa cells. PMID : 9006934. The nuclear counter stain is DAPI (blue).

Tubulin is detected with Anti-alpha Tubulin mouse MAb (ab7291) at 1/1000 dilution, followed by Goat Anti-Mouse IgG H&L (Alexa Fluor^® 594) (ab150120) secondary antibody at 1/1000 dilution (red).

The negative controls are as follows :

-ve control 1 : ab207447 at 1/500 dilution, followed by Goat Anti-Mouse IgG H&L (Alexa Fluor^® 594) (ab150120) secondary at 1/1000 dilution.

-ve control 2 : Anti-alpha Tubulin mouse MAb (ab7291) at 1/1000 dilution, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) secondary at 1/1000 dilution.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab207447).

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-Smad2 + Smad3 antibody [EPR19557] - BSA and Azide free (AB236030)

Intracellular flow cytometric analysis of 4% paraformaldehyde-fixed HeLa (Human epithelial cell line from cervix adenocarcinoma) cells labeling Smad2 and Smad3 with ab207447 at 1/500 dilution (red) compared with a rabbit monoclonal IgG isotype control (ab172730; black) and an unlabelled control (cells without incubation with primary antibody and secondary antibody; blue). Goat anti rabbit IgG (Alexa Fluor^® 488) at 1/500 dilution was used as the secondary antibody.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab207447).

• IP

Lab

Immunoprecipitation - Anti-Smad2 + Smad3 antibody [EPR19557] - BSA and Azide free (AB236030)

ab207447 Immunoprecipitating Smad2 + Smad3 in human HeLa whole cell lysate . 2μg of capture antibody in 0.35mg lysate was used. 10μg of cell lysate was incubated with primary antibody at a dilution of 1/1000 and VeriBlot for IP Detection Reagent (HRP) ab131366 was used for detection at a dilution of 1/1000.

Lane 1 : HeLa (human cervix adenocarcinoma) whole cell lysate
Lane 2 : HeLa (human cervix adenocarcinoma) whole cell lysate
Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab207447 in HeLa (human cervix adenocarcinoma) whole cell lysate

Blocking buffer : 5% NFDM/TBST

Diluting buffer : 5% NFDM/TBST

Smad2 and Smad3 can be resolved using a lower percentage gel.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab207447).

All lanes:

Immunoprecipitation - Anti-Smad2 + Smad3 antibody [EPR19557] - ChIP Grade (<a href='/en-us/products/primary-antibodies/smad2-smad3-antibody-epr19557-chip-grade-ab207447'>ab207447</a>)

Observed band size: 58-62 kDa

false

Exposure time: 3min

• IP

Supplier Data

Immunoprecipitation - Anti-Smad2 + Smad3 antibody [EPR19557] - BSA and Azide free (AB236030)

Smad2 + Smad3 were immunoprecipitated from 1mg of HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate with ab207447 at 1/40 dilution. Western blot was performed from the immunoprecipitate using ab207447 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 : ab207447 IP in HeLa whole cell lysate.

Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab207447 in HeLa whole cell lysate.

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

Exposure time : 5 seconds.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab207447).

All lanes:

Immunoprecipitation - Anti-Smad2 + Smad3 antibody [EPR19557] - ChIP Grade (<a href='/en-us/products/primary-antibodies/smad2-smad3-antibody-epr19557-chip-grade-ab207447'>ab207447</a>)

false

• ChIP

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ChIP - Anti-Smad2 + Smad3 antibody [EPR19557] - BSA and Azide free (AB236030)

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 ab207447 (blue), and 20µl of Anti rabit IgG sepharose beads. 2μg of rabbit normal IgG was added to the beads control (yellow). The immunoprecipitated DNA was quantified by real time PCR (Sybr green approach).

The ChIP condition is designed against Smad2 refer to PMID : 18955504.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab207447).

• ChIC/CUT&RUN-seq

Lab

ChIC/CUT&RUN sequencing - Anti-Smad2 + Smad3 antibody [EPR19557] - BSA and Azide free (AB236030)

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab207447).

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 ab207447 [EPR19557]. 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.