Anti-Smad4 antibody [SP306] - C-terminal

• ICC/IF

Unknown

Immunocytochemistry/ Immunofluorescence - Anti-Smad4 antibody [SP306] - C-terminal (AB217267)

Immunocytochemistry/ Immunofluorescence analysis of HepG2 (human hepatocellular carcinoma epithelial cell) cells labeling Smad4 with purified ab217267 at 1 : 50 (9 µg/ml). Cells were fixed in 4% paraformaldehyde and permeabilized with 0.1% Triton X-100. Cells were counterstained with ab195889 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor® 594) 1 : 200 (2.5 µg/ml). Goat anti rabbit IgG (Alexa Fluor® 488, ab150077) was used as the secondary antibody at 1 : 1000 (2 µg/ml) dilution. DAPI nuclear counterstain. PBS instead of the primary antibody was used as the secondary antibody only control.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Smad4 antibody [SP306] - C-terminal (AB217267)

Immunohistochemical analysis of formalin-fixed, paraffin-embedded human colon adenocarcinoma tissue labeling Smad4 with ab217267 at 1/100 dilution.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Smad4 antibody [SP306] - C-terminal (AB217267)

Immunohistochemical analysis of formalin-fixed, paraffin-embedded human renal cell carcinoma tissue labeling Smad4 with ab217267 at 1/100 dilution.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Smad4 antibody [SP306] - C-terminal (AB217267)

Immunohistochemical analysis of formalin-fixed, paraffin-embedded human placenta tissue labeling Smad4 with ab217267 at 1/100 dilution.

• Flow Cyt (Intra)

Lab

Flow Cytometry (Intracellular) - Anti-Smad4 antibody [SP306] - C-terminal (AB217267)

Flow cytometry overlay histogram showing wild-type Hap1 (green line) and SMAD4 knockout Hap1 stained with ab217267 (red line). The cells were fixed with 80% methanol (5 min) and then permeabilised with 0.1% PBS-Triton X-100 for 15 min. The cells were then incubated in 1x PBS containing 10% normal goat serum to block non-specific protein-protein interaction followed by the antibody (ab217267) (1x 106 in 100μl at 0.008 μg/ml (1/265000)) for 30min at 22°C.

The secondary antibody Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed was incubated at 1/4000 for 30min at 22°C

Isotype control antibody Recombinant Rabbit IgG, monoclonal [EPR25A] - Isotype Control was used at the same concentration and conditions as the primary antibody (wild-type Hap1 - black line, SMAD4 knockout Hap1 - grey line). Unlabelled sample was also used as a control (this line is not shown for the purpose of simplicity).

Acquisition of >5000 events were collected using a 50 mW Blue laser (488nm) and 525/40 bandpass filter.

This antibody gave a positive signal in Hap1 Fixed with 4% formaldehyde (10 min) / permeabilised with 0.1% PBS-Triton X-100 for 15 min under the same conditions.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Smad4 antibody [SP306] - C-terminal (AB217267)

Immunohistochemical analysis of formalin-fixed, paraffin-embedded human pancreatic adenocarcinoma tissue labeling Smad4 with ab217267 at 1/100 dilution.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Smad4 antibody [SP306] - C-terminal (AB217267)

Immunohistochemical analysis of formalin-fixed, paraffin-embedded human colon tissue labeling Smad4 with ab217267 at 1/100 dilution.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Smad4 antibody [SP306] - C-terminal (AB217267)

Immunohistochemical analysis of formalin-fixed, paraffin-embedded human kidney tissue labeling Smad4 with ab217267 at 1/100 dilution.

• Flow Cyt (Intra)

Unknown

Flow Cytometry (Intracellular) - Anti-Smad4 antibody [SP306] - C-terminal (AB217267)

Intracellular Flow Cytometry analysis of HepG2 (Human hepatocellular carcinoma epithelial cell) cells labeling Smad4 with purified ab217267 at 1/450 dilution (1.01 μg/ml) Red. Cells were fixed with 4% paraformaldehyde . A Goat anti rabbit IgG (Alexa Fluor^® 488, ab150077) secondary antibody was used at 1/2000 dilution. Isotype control - Rabbit monoclonal IgG (ab172730) / Black. Unlabeled control - Unlabelled cells / Blue.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Smad4 antibody [SP306] - C-terminal (AB217267)

Immunohistochemical analysis of formalin-fixed, paraffin-embedded human pancreas tissue labeling Smad4 with ab217267 at 1/100 dilution.

• Flow Cyt (Intra)

Unknown

Flow Cytometry (Intracellular) - Anti-Smad4 antibody [SP306] - C-terminal (AB217267)

Intracellular Flow Cytometry analysis of NIH/3T3 (Mouse embryonic fibroblast) cells labeling Smad4 with purified ab217267 at 1/450 dilution (1.01 μg/ml) Red. Cells were fixed with 4% paraformaldehyde . A Goat anti rabbit IgG (Alexa Fluor^® 488, ab150077) secondary antibody was used at 1/2000 dilution. Isotype control - Rabbit monoclonal IgG (ab172730) / Black. Unlabeled control - Unlabelled cells / Blue.

• Flow Cyt (Intra)

Supplier Data

Flow Cytometry (Intracellular) - Anti-Smad4 antibody [SP306] - C-terminal (AB217267)

Intracellular flow cytometric analysis of NIH/3T3 (Mouse embryo fibroblast cell line) cellslabeling Smad4 with ab217267 at 1/400 dilution for 30 minutes at 4° C (green), compared to a negative control cell of rabbit IgG (blue).

• Flow Cyt (Intra)

Unknown

Flow Cytometry (Intracellular) - Anti-Smad4 antibody [SP306] - C-terminal (AB217267)

Intracellular Flow Cytometry analysis of C6 (Rat glial tumor glial cell) cells labeling Smad4 with purified ab217267 at 1/450 dilution (1.01 μg/ml) Red. Cells were fixed with 4% paraformaldehyde . A Goat anti rabbit IgG (Alexa Fluor^® 488, ab150077) secondary antibody was used at 1/2000 dilution. Isotype control - Rabbit monoclonal IgG (ab172730) / Black. Unlabeled control - Unlabelled cells / Blue.

• ICC/IF

Unknown

Immunocytochemistry/ Immunofluorescence - Anti-Smad4 antibody [SP306] - C-terminal (AB217267)

Immunocytochemistry/ Immunofluorescence analysis of C6 (rat glial tumor glial cell) cells labeling Smad4 with purified ab217267 at 1 : 50 (9 µg/ml). Cells were fixed in 4% paraformaldehyde and permeabilized with 0.1% Triton X-100. Cells were counterstained with ab195889 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor® 594) 1 : 200 (2.5 µg/ml). Goat anti rabbit IgG (Alexa Fluor® 488, ab150077) was used as the secondary antibody at 1 : 1000 (2 µg/ml) dilution. DAPI nuclear counterstain. PBS instead of the primary antibody was used as the secondary antibody only control.

• ICC/IF

Unknown

Immunocytochemistry/ Immunofluorescence - Anti-Smad4 antibody [SP306] - C-terminal (AB217267)

Immunocytochemistry/ Immunofluorescence analysis of NIH/3T3 (mouse embryonic fibroblast) cells labeling Smad4 with purified ab217267 at 1 : 50 (9 µg/ml). Cells were fixed in 4% paraformaldehyde and permeabilized with 0.1% Triton X-100. Cells were counterstained with ab195889 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor® 594) 1 : 200 (2.5 µg/ml). Goat anti rabbit IgG (Alexa Fluor® 488, ab150077) was used as the secondary antibody at 1 : 1000 (2 µg/ml) dilution. DAPI nuclear counterstain. PBS instead of the primary antibody was used as the secondary antibody only control.

• ChIC/CUT&RUN-seq

Lab

ChIC/CUT&RUN sequencing - Anti-Smad4 antibody [SP306] - C-terminal (AB217267)

ChIC/CUT&RUN was performed using a pAG-MNase at a final concentration of 700 ng/mL, 2.5 x 10⁵ HeLa (Human epithelial cell line from cervix adenocarcinoma) cells treated with hTGF-β1 (10 ng/mL 1 h) and 5 µg of ab217267 [SP306]. 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-Smad4 antibody [SP306] - C-terminal (AB217267)

ChIC/CUT&RUN was performed using a pAG-MNase at a final concentration of 700 ng/mL, 2.5 x 10⁵ HeLa (Human epithelial cell line from cervix adenocarcinoma) cells treated with hTGF-β1 (10 ng/mL 1 h) and 5 µg of ab217267 [SP306]. 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-Smad4 antibody [SP306] - C-terminal (AB217267)

ChIC/CUT&RUN was performed using a pAG-MNase at a final concentration of 700 ng/mL, 2.5 x 10⁵ HeLa (Human epithelial cell line from cervix adenocarcinoma) cells treated with hTGF-β1 (10 ng/mL 1 h) and 5 µg of ab217267 [SP306]. 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.