All lanes : Anti-Smad3 antibody [EPR19686] - ChIP Grade (ab208182) at 1/1000 dilution

Lane 1 : Wild-type A549 cell lysate
Lane 2 : SMAD3 knockout A549 cell lysate
Lane 3 : HeLa cell lysate
Lane 4 : Human Kidney cell lysate

Lysates/proteins at 20 µg per lane.

Performed under reducing conditions.

Predicted band size: 48 kDa
Observed band size: 50 kDa why is the actual band size different from the predicted?

Lanes 1 - 4: Merged signal (red and green). Green - ab208182 observed at 50 kDa. Red - loading control, ab8245 (Mouse anti-GAPDH antibody [6C5]) observed at 37kDa.

ab208182 was shown to react with Smad3 in western blot. Membranes were blocked in 3% milk in TBS-T (0.1% Tween^®) before incubation with ab208182 and ab8245 (Mouse anti-GAPDH antibody [6C5]) overnight at 4°C at a 1 in 1000 Dilution and a 1 in 20000 dilution respectively. Blots were incubated with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preabsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed (ab216776) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.

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 ab208182 (red), and 20µl of protein A/G beads. 2μg of rabbit normal IgG was added to the beads control (grey). The immunoprecipitated DNA was quantified by real time PCR (Sybr green approach).

The ChIP condition performed here is similar to the literature (PMID: 18245174).

All lanes : Anti-Smad3 antibody [EPR19686] - ChIP Grade (ab208182) at 1/1000 dilution

Lane 1 : Wild-type HeLa cell lysate
Lanes 2 & 4 : SMAD3 knockout HeLa cell lysate
Lane 3 : Wild-type HeLa cell lysate

Lysates/proteins at 20 µg per lane.

Performed under reducing conditions.

Predicted band size: 48 kDa
Observed band size: 74 kDa why is the actual band size different from the predicted?

Lanes 1 - 4: Merged signal (red and green). Green - ab208182 observed at 55 kDa. Red - loading control, ab8245 observed at 37 kDa.  

 ab208182 was shown to react with Smad3 in wild-type HeLa cells. Loss of signal was observed when knockout cell line ab255431 (knockout cell lysate ab263834) was used. Wild-type and Smad3 knockout samples were subjected to SDS-PAGE. ab208182 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) were incubated overnight at 4°C at 1 in 1000 dilution and 1 in 20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preadsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preadsorbed (ab216776) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.

 

All lanes : Anti-Smad3 antibody [EPR19686] - ChIP Grade (ab208182) at 1/1000 dilution

Lane 1 : Human Smad3 recombinant protein
Lane 2 : Human Smad2 active protein

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: 48 kDa
Observed band size: 74 kDa why is the actual band size different from the predicted?


Exposure time: 1 second

Blocking/Dilution buffer: 5% NFDM/TBST.

Human Smad3 recombinant protein contains aa2-227 with GST and His-Tag®. This protein was made-in house.

All lanes : Anti-Smad3 antibody [EPR19686] - ChIP Grade (ab208182) at 1/1000 dilution

Lane 1 : HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate
Lane 2 : Jurkat (Human T cell leukemia cell line from peripheral blood) whole cell lysate
Lane 3 : HEK-293 (Human epithelial cell line from embryonic kidney) whole cell lysate
Lane 4 : K562 (Human chronic myelogenous leukemia cell line from bone marrow) whole cell lysate
Lane 5 : BxPC-3 (Human pancreas adenocarcinoma cell line) whole cell lysate
Lane 6 : Human fetal kidney lysate

Lysates/proteins at 10 µg per lane.

Secondary
Lanes 1-5 : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution
Lane 6 : Goat Anti-Rabbit IgG Peroxidase Conjugate, specific to the non-reduced form of IgG at 1/10000 dilution

Predicted band size: 48 kDa
Observed band size: 55 kDa why is the actual band size different from the predicted?

Blocking/Dilution buffer: 5% NFDM/TBST.

Exposure time: Lane 1/6: 3 minutes; Lane 2-5: 15 seconds.

All lanes : Anti-Smad3 antibody [EPR19686] - ChIP Grade (ab208182) at 1/1000 dilution

Lane 1 : C6 (Rat glial tumor cell line) whole cell lysate
Lane 2 : PC-12 (Rat adrenal gland pheochromocytoma cell line) whole cell lysate
Lane 3 : NIH/3T3 (Mouse embryonic fibroblast cell line) whole cell lysate
Lane 4 : Rat spleen lysate
Lane 5 : Rat kidney lysate
Lane 6 : Mouse 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: 48 kDa
Observed band size: 55 kDa why is the actual band size different from the predicted?

Blocking/Dilution buffer: 5% NFDM/TBST.

Exposure time: Lane 1/2: 30 seconds; Lane 3-6: 3 minutes.

Smad3 was immunoprecipitated from 0.35 mg of HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate with ab208182 at 1/30 dilution.

Western blot was performed from the immunoprecipitate using ab208182 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: ab208182 IP in HeLa whole cell lysate.

Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab208182 in HeLa whole cell lysate.

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

Exposure time: 3 minutes.