Anti-Smad4 antibody [EPR22589-112] - BSA and Azide free

• ICC/IF

Unknown

Immunocytochemistry/ Immunofluorescence - Anti-Smad4 antibody [EPR22589-112] - BSA and Azide free (AB254290)

Immunofluorescent analysis of HeLa (human cervix adenocarcinoma epithelial cell) cells labeling Smad4 with ab230815 at a 1/100 dilution (green). Cells were fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100. Confocal image showing mainly nuclear staining in HeLa cell line treated with TGF-beta (10ng/ml) for 1 h. AlexaFluor^®488 Goat anti-Rabbit (ab150077) was used secondary antibody at 1/1000 dilution, this was also used on its own as a control. DAPI was used as a nuclear counterstain (blue). Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594, ab195889) was used as a counterstain at 1/200 dilution.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab230815).

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-Smad4 antibody [EPR22589-112] - BSA and Azide free (AB254290)

Intracellular flow cytometric analysis of 4% paraformaldehyde-fixed Smad4 KO HAP1 (Smad4 knockout human chronic myelogenous leukemia near-haploid cell line, Left) / WT HAP1 (human chronic myelogenous leukemia near-haploid cell line, Right) cell line labeling Smad4 with ab230815 at 1/500 (Red) compared with a Rabbit monoclonal IgG (ab172730) (Black) and an unlabeled control (cells without incubation with primary antibody and secondary antibody) (Blue). Goat anti rabbit IgG (Alexa Fluor^® 488, ab150077) was used as secondary antibody at 1/2000 dilution.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab230815).

• IP

Unknown

Immunoprecipitation - Anti-Smad4 antibody [EPR22589-112] - BSA and Azide free (AB254290)

Smad4 was immunoprecipitated from 0.35mg of HeLa (human cervix adenocarcinoma epithelial cell) whole cell lysate with ab230815 at 1/30 dilution. Western Blot was performed from the immunoprecipitate using ab230815 at 1/1000 dilution (0.5 μg/ml).

VeriBlot for IP Detection Reagent (HRP) (ab131366), was used as secondary antibody at 1/5000 dilution.

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

Lane 2 : ab230815 IP in HeLa whole cell lysate

Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab230815 in HeLa whole cell lysate

Blocking/Dilution buffer : 5% NFDM/TBST.

Exposure time : 30 seconds

Lysate was freshly prepared and IP test was done immediately to avoid protein degradation. Lysate incubation time was shortened from overnight to 2h.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab230815).

All lanes:

Immunoprecipitation - Anti-Smad4 antibody [EPR22589-112] (<a href='/en-us/products/primary-antibodies/smad4-antibody-epr22589-112-ab230815'>ab230815</a>)

Predicted band size: 60 kDa

false

• IP

Unknown

Immunoprecipitation - Anti-Smad4 antibody [EPR22589-112] - BSA and Azide free (AB254290)

Smad4 was immunoprecipitated from 0.35mg of NIH/3T3 (mouse embryonic fibroblast) whole cell lysate with ab230815 at 1/30 dilution. Western Blot was performed from the immunoprecipitate using ab230815 at 1/1000 dilution (0.5 μg/ml).

VeriBlot for IP Detection Reagent (HRP) (ab131366), was used as secondary antibody at 1/5000 dilution.

Lane 1 : NIH/3T3 whole cell lysate 10μg (Input).

Lane 2 : ab230815 IP in NIH/3T3 whole cell lysate

Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab230815 in NIH/3T3 whole cell lysate

Blocking/Dilution buffer : 5% NFDM/TBST.

Exposure time : 10 seconds

Lysate was freshly prepared and IP test was done immediately to avoid protein degradation. Lysate incubation time was shortened from overnight to 2h.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab230815).

All lanes:

Immunoprecipitation - Anti-Smad4 antibody [EPR22589-112] (<a href='/en-us/products/primary-antibodies/smad4-antibody-epr22589-112-ab230815'>ab230815</a>)

Predicted band size: 60 kDa

false

• WB

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Western blot - Anti-Smad4 antibody [EPR22589-112] - BSA and Azide free (AB254290)

Blocking/Dilution buffer : 5% NFDM/TBST.

Exposure Time : Lanes 1-6 : 15 secs; Lanes 7-8 : 114 secs; Lane 9 : 3 mins.

ab230815 was shown to specifically react with Smad4 in wild-type HAP1 cells as signal was lost in Smad4 knockout cells. Wild-type and Smad4 knockout samples were subjected to SDS-PAGE. ab230815 and ab181602 (Rabbit anti-GAPDH loading control) were incubated 1 hour at room temperature at 1/1000 dilution and 1/200,000 dilution respectively. Blots were developed with Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (ab97051) secondary antibody at 1/100,000 dilution for 1 hour at room temperature before imaging.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab230815).

All lanes:

Western blot - Anti-Smad4 antibody [EPR22589-112] (<a href='/en-us/products/primary-antibodies/smad4-antibody-epr22589-112-ab230815'>ab230815</a>) at 1/1000 dilution

Lane 1:

Smad4 knockout HAP1 whole cell lysate at 20 µg

Lane 2:

Wild-type HAP1 whole cell lysate at 20 µg

Lane 3:

HeLa (human cervix adenocarcinoma epithelial cell), whole cell lysate at 20 µg

Lane 4:

HCT116 (human colorectal carcinoma epithelial cell) whole cell lysate at 20 µg

Lane 5:

HepG2 (human hepatocellular carcinoma epithelial cell) whole cell lysate at 20 µg

Lane 6:

Human fetal lung tissue lysate at 20 µg

Lane 7:

Mouse embryo tissue lysate at 20 µg

Lane 8:

Mouse lung tissue lysate at 20 µg

Lane 9:

Rat lung tissue lysate at 20 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: 60 kDa

Observed band size: 60 kDa

false

• ChIC/CUT&RUN-seq

Lab

ChIC/CUT&RUN sequencing - Anti-Smad4 antibody [EPR22589-112] - BSA and Azide free (AB254290)

This data was developed using the same antibody clone in a different buffer formulation (ab230815).

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 ab230815 [EPR22589-112]. 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 [EPR22589-112] - BSA and Azide free (AB254290)

This data was developed using the same antibody clone in a different buffer formulation (ab230815).

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 ab230815 [EPR22589-112]. 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 [EPR22589-112] - BSA and Azide free (AB254290)

This data was developed using the same antibody clone in a different buffer formulation (ab230815).

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 ab230815 [EPR22589-112]. 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.