All lanes : Anti-Smad2 + Smad3 antibody [EPR19557] - ChIP Grade (ab207447) at 1/50000 dilution

Lane 1 : Recombinant protein fragment human Smad2
Lane 2 : Recombinant protein fragment human Smad3

Lysates/proteins at 0.01 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

Predicted band size: 52 kDa
Observed band size: 38,60 kDa why is the actual band size different from the predicted?

Blocking/Dilution buffer: 5% NFDM/TBST.

Exposure time: Lane 1: 2 seconds; Lane 2: 15 seconds.

Human Smad2 fragment recombinant protein contains aa2-270 with His-Tag^®. Human Smad3 fragment recombinant protein contains aa2-227 with His-Tag^® and GST-tag.

All lanes : Anti-Smad2 + Smad3 antibody [EPR19557] - ChIP Grade (ab207447) at 1/10000 dilution

Lane 1 : HeLa (Human T cell leukemia cell line from peripheral blood) whole cell lysate
Lane 2 : K562 (Human chronic myelogenous leukemia cell line from bone marrow) whole cell lysate
Lane 3 : A549 (Human lung carcinoma cell line) whole cell lysate
Lane 4 : Human fetal liver lysate

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

Predicted band size: 52 kDa
Observed band size: 58-62 kDa why is the actual band size different from the predicted?


Exposure time: 3 minutes

Blocking/Dilution buffer: 5% NFDM/TBST.

Anti-Smad2 + Smad3 antibody [EPR19557] - ChIP Grade (ab207447) at 1/2000 dilution + Jurkat (Human T cell leukemia cell line from peripheral blood) whole cell lysate at 10 µg

Secondary
Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

Predicted band size: 52 kDa
Observed band size: 58-62 kDa why is the actual band size different from the predicted?


Exposure time: 15 seconds

Blocking/Dilution buffer: 5% NFDM/TBST.

Anti-Smad2 + Smad3 antibody [EPR19557] - ChIP Grade (ab207447) at 1/10000 dilution + HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate at 10 µg

Secondary
Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

Predicted band size: 52 kDa
Observed band size: 52,58-62 kDa why is the actual band size different from the predicted?


Exposure time: 30 seconds

Blocking/Dilution buffer: 5% NFDM/TBST.

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

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.

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.

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.

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.

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.