All lanes : Anti-Smad2 + Smad3 antibody [EPR19557-4] - ChIP Grade (ab202445) at 1/1000 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?


Exposure time: 1 second

Blocking/Dilution buffer: 5% NFDM/TBST.

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 [EPR19557-4] - ChIP Grade (ab202445) at 1/2000 dilution

Lane 1 : 293 (Human epithelial cell line from embryonic kidney) whole cell lysate
Lane 2 : HepG2 (Human liver hepatocellular carcinoma cell line) whole cell lysate
Lane 3 : HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate
Lane 4 : Jurkat (Human T cell leukemia cell line from peripheral blood) whole cell 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?

Blocking/Dilution buffer: 5% NFDM/TBST.

Exposure time: Lane 1/2: 10 seconds; Lane 3: 3 seconds; Lane 4: 1 seconds.

All lanes : Anti-Smad2 + Smad3 antibody [EPR19557-4] - ChIP Grade (ab202445) at 1/2000 dilution

Lane 1 : Human fetal heart lysate
Lane 2 : Human fetal kidney lysate

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG Peroxidase Conjugate, specific to the non-reduced form of IgG at 1/10000 dilution

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

Blocking/Dilution buffer: 5% NFDM/TBST.

Exposure time: Lane 1: 30 seconds; Lane 2: 10 seconds.

Lane 1 : Anti-Smad2 + Smad3 antibody [EPR19557-4] - ChIP Grade (ab202445) at 1/1000 dilution
Lanes 2 & 5 : Anti-Smad2 + Smad3 antibody [EPR19557-4] - ChIP Grade (ab202445) at 1/2000 dilution
Lanes 3-4 : Anti-Smad2 + Smad3 antibody [EPR19557-4] - ChIP Grade (ab202445) at 1/20000 dilution

Lane 1 : Mouse brain lysate
Lane 2 : Mouse heart lysate
Lane 3 : Rat brain lysate
Lane 4 : C6 (Rat glial tumor cell line) whole cell lysate
Lane 5 : Rat spleen 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?

Blocking/Dilution buffer: 5% NFDM/TBST.

Exposure time: Lane 1: 3 minutes; Lane 2: 10 seconds; Lane 3: 30 seconds; Lane 4: 3 seconds; Lane 5: 5 seconds.

 

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 ab202445 at 1/200 dilution, followed by Goat Anti-Rabbit IgG (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution (green). The results show signal translocation after TGF-beta (10ng/ml, 1h) treatment on HeLa cells.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: ab202445 at 1/200 dilution, followed by Goat Anti-Mouse IgG H&L (Alexa Fluor^® 594) (ab150120) secondary antibody 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 antibody at 1/1000 dilution.

Flow cytometric analysis of 4% paraformaldehyde-fixed HeLa (Human epithelial cell line from cervix adenocarcinoma) cells labeling Smad2 + Smad3 with ab202445 at 1/600 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 1/2000 dilution was used as the secondary antibody.

Smad2 + Smad3 was immunoprecipitated from 0.35mg of HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate with ab202445 at 1/40 dilution. Western blot was performed from the immunoprecipitate using ab202445 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: ab202445 IP in HeLa whole cell lysate.

Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab202445 in HeLa whole cell lysate.

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

Exposure time: 3 seconds.

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 ab202445 (blue), and 20µl of Anti rabbit 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).