All lanes : Anti-Smad3 antibody (ab84177) at 1 µg/ml

Lane 1 : Wild-type A549 cell lysate
Lane 2 : SMAD3 knockout A549 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 - 2: Merged signal (red and green). Green - ab84177 observed at 50 kDa. Red - loading control ab8245 (Mouse anti-GAPDH antibody [6C5]) observed at 37kDa.

ab84177 was shown to react with Smad3 in wild-type A549 cells in western blot with loss of signal observed in SMAD3 knockout sample. Wild-type and SMAD3 knockout A549 cell lysates were subjected to SDS-PAGE. Membranes were blocked in 3% milk in TBS-T (0.1% Tween^®) before incubation with ab84177 and ab8245 (Mouse anti-GAPDH antibody [6C5]) overnight at 4°C at 1 µg/ml 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.

Smad3 was immunoprecipitated using 0.5mg Hela whole cell extract, 5µg of Rabbit polyclonal to Smad3 and 50µl of protein G magnetic beads (+). No antibody was added to the control (-).
The antibody was incubated under agitation with Protein G beads for 10min, Hela whole cell extract lysate diluted in RIPA buffer was added to each sample and incubated for a further 10min under agitation.
Proteins were eluted by addition of 40µl SDS loading buffer and incubated for 10min at 70^oC; 10µl of each sample was separated on a SDS PAGE gel, transferred to a nitrocellulose membrane, blocked with 5% BSA and probed with ab84177.
Secondary: Mouse monoclonal [SB62a] Secondary Antibody to Rabbit IgG light chain (HRP) (ab99697).
Band: 50kDa: Smad3

ICC/IF image of ab84177 stained HepG2 cells. The cells were 4% PFA fixed (10 min) and then incubated in 1%BSA / 10% normal Goat serum / 0.3M glycine in 0.1% PBS-Tween for 1h to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab84177, 5µg/ml) overnight at +4°C. The secondary antibody (green) was Alexa Fluor® 488 Goat anti-Rabbit IgG (H+L) used at a 1/1000 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (red) at a 1/200 dilution for 1h. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43µM. This antibody also gave a positive result in 4% PFA fixed (10 min) HeLa cells at 5µg/ml.

All lanes : Anti-Smad3 antibody (ab84177) at 1 µg/ml

Lane 1 : A431 (Human epithelial carcinoma cell line) Whole Cell Lysate
Lane 2 : EGF-treated A431 (Human epithelial carcinoma cell line) Whole Cell Lysate

Lysates/proteins at 10 µg/ml per lane.

Secondary
All lanes : Goat polyclonal to Rabbit IgG - H&L - Pre-Adsorbed (HRP) at 1/3000 dilution

Developed using the ECL technique.

Performed under reducing conditions.

Predicted band size: 48 kDa
Observed band size: 52 kDa why is the actual band size different from the predicted?


Exposure time: 20 minutes

IHC image of Ab84177 staining in Human Cervical carcinoma formalin fixed paraffin embedded tissue section, performed on a Leica Bond^TM system using the standard protocol F. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20 mins. The section was then incubated with Ab84177, 1µg/ml, for 15 mins at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX.

For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.

All lanes : Anti-Smad3 antibody (ab84177) at 1 µg/ml

Lane 1 : HeLa (Human epithelial carcinoma cell line) Nuclear Lysate
Lane 2 : HeLa (Human epithelial carcinoma cell line) Whole Cell Lysate
Lane 3 : NIH 3T3 (Mouse embryonic fibroblast cell line) Whole Cell Lysate

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : Goat polyclonal Secondary Antibody to Rabbit IgG - H&L (HRP), pre-adsorbed at 1/5000 dilution

Developed using the ECL technique.

Performed under reducing conditions.

Predicted band size: 48 kDa
Observed band size: 48 kDa
Additional bands at: 37 kDa, 50 kDa. We are unsure as to the identity of these extra bands.


Exposure time: 8 minutes