Anti-SMAD5 antibody [EP619Y]

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-SMAD5 antibody [EP619Y] (AB40771)

Immunohistochemical staining of paraffin embedded human testis with purified ab40771 at a working dilution of 1/50. The secondary antibody used is ab97051, a goat anti-rabbit IgG (H&L) at a dilution of 1/500. The sample is counter-stained with hematoxylin. Antigen retrieval was perfomed using Tris-EDTA buffer, pH 9.0. PBS was used instead of the primary antibody as the negative control, and is shown in the inset.

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-SMAD5 antibody [EP619Y] (AB40771)

Immunofluorescence staining of HeLa cells with purified ab40771 at a working dilution of 1/100, counter-stained with DAPI. The secondary antibody was Alexa Fluor^® 488 goat anti-rabbit (ab150077), used at a dilution of 1/1000. ab7291, a mouse anti-tubulin antibody (1/1000), was used to stain tubulin along with ab150120 (Alexa Fluor^® 594 goat anti-mouse, 1/1000), shown in the top right hand panel. The cells were fixed in 4% PFA and permeabilized using 0.1% Triton X 100. The negative controls are shown in bottom middle and right hand panels - for negative control 1, purified ab40771 was used at a dilution of 1/500 followed by an Alexa Fluor^® 594 goat anti-mouse antibody (ab150120) at a dilution of 1/500. For negative control 2, ab7291 (mouse anti-tubulin) was used at a dilution of 1/500 followed by an Alexa Fluor^® 488 goat anti-rabbit antibody (ab150077) at a dilution of 1/400.

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-SMAD5 antibody [EP619Y] (AB40771)

Overlay histogram showing HEK293 cells stained with unpurified ab40771 (red line). The cells were fixed with methanol (5 min) and then permeabilized with 0.1% PBS-Tween for 20 min. The cells were then incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions followed by the antibody (ab40771, 1/50 dilution) for 30 min at 22°C. The secondary antibody used was DyLight^® 488 goat anti-rabbit IgG (H+L) (ab96899) at 1/500 dilution for 30 min at 22°C. Isotype control antibody (black line) was rabbit monoclonal IgG (1μg/1x10⁶ cells) used under the same conditions. Acquisition of >5,000 events was performed. This antibody gave a positive signal in HEK293 cells fixed with 4% paraformaldehyde (10 min)/permeabilized in 0.1% PBS-Tween used under the same conditions.

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-SMAD5 antibody [EP619Y] (AB40771)

Unpurified ab40771 (4μg/ml) staining SMAD5 in human skin using an automated system (DAKO Autostainer Plus). Using this protocol there is strong staining of nuclear/cytoplasmic compartments within the stratum granulosum.
Sections were rehydrated and antigen retrieved with the Dako 3 in 1 AR buffer EDTA pH 9.0 in a DAKO PT link. Slides were peroxidase blocked in 3% H2O2 in methanol for 10 mins. They were then blocked with Dako Protein block for 10 minutes (containing casein 0.25% in PBS) then incubated with primary antibody for 20 min and detected with Dako envision flex amplification kit for 30 minutes. Colorimetric detection was completed with Diaminobenzidine for 5 minutes. Slides were counterstained with Haematoxylin and coverslipped under DePeX. Please note that, for manual staining, optimization of primary antibody concentration and incubation time is recommended. Signal amplification may be required.

• Flow Cyt (Intra)

Unknown

Flow Cytometry (Intracellular) - Anti-SMAD5 antibody [EP619Y] (AB40771)

Overlay histogram showing PC-12 cells fixed in 4% PFA and stained with purified ab40771 at a dilution of 1/100 (red line). The secondary antibody used was Alexa Fluorr^® 488 goat anti-rabbit at a dilution of 1/500. Rabbit monoclonal IgG was used as an isotype control (black line) and cells incubated in the absence of both primary and secondary antibody were used as a negative control (blue line).

• WB

Lab

Western blot - Anti-SMAD5 antibody [EP619Y] (AB40771)

Blocking buffer : 5% NFDM/TBST
Dilution buffer : 5% NFDM/TBST

All lanes:

Western blot - Anti-SMAD5 antibody [EP619Y] (ab40771) at 1/5000 dilution

Lane 1:

HEK293 whole cell lysate at 10 µg

Lane 2:

COS-1 whole cell lysate at 10 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 1/20000 dilution

Predicted band size: 52 kDa

Observed band size: 52 kDa

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• WB

Lab

Western blot - Anti-SMAD5 antibody [EP619Y] (AB40771)

Anti-SMAD5 antibody [EP619Y] (ab40771) staining at 1/1000 dilution, shown in green; Mouse anti-CANX [CANX/1543] (ab238078) loading control staining at 1/20000 dilution, shown in magenta. In Western blot, ab40771 was shown to bind specifically to SMAD5. A band was observed at 52 kDa in wild-type HeLa cell lysates with no signal observed at this size in SMAD5 knockout cell line. To generate this image, wild-type and SMAD5 knockout HeLa cell lysates were analysed. First, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 3 % milk in TBS-0.1 % Tween^® 20 (TBS-T) before incubation with primary antibodies overnight at 4 °C. Blots were washed four times in TBS-T, incubated with secondary antibodies for 1 h at room temperature, washed again four times then imaged. Secondary antibodies used were Goat anti-Rabbit IgG H&L 800CW and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution.

All lanes:

Western blot - Anti-SMAD5 antibody [EP619Y] (ab40771) at 1/1000 dilution

Lane 1:

Wild-type HeLa Vehicle Control TGF-beta (0 ng/mL, 30 min) cell lysate at 20 µg

Lane 2:

Wild-type HeLa Treated TGF-beta (10 ng/mL, 30 min) cell lysate at 20 µg

Lane 3:

SMAD1 knockout HeLa Vehicle Control TGF-beta (0 ng/mL, 30 min), <a href='/en-us/products/cell-lines/human-smad1-knockout-hela-cell-line-ab265400'>ab265400</a> cell lysate at 20 µg

Lane 4:

SMAD1 knockout HeLa Treated TGF-beta (10 ng/mL, 30 min), <a href='/en-us/products/cell-lines/human-smad1-knockout-hela-cell-line-ab265400'>ab265400</a> cell lysate at 20 µg

Lane 5:

SMAD2 knockout HeLa Vehicle Control TGF-beta (0 ng/mL, 30 min), <a href='/en-us/products/cell-lines/human-smad2-knockout-hela-cell-line-ab255430'>ab255430</a> cell lysate at 20 µg

Lane 6:

SMAD2 knockout HeLa Treated TGF-beta (10 ng/mL, 30 min), <a href='/en-us/products/cell-lines/human-smad2-knockout-hela-cell-line-ab255430'>ab255430</a> cell lysate at 20 µg

Lane 7:

Wild-type HeLa Vehicle Control TGF-beta (0 ng/mL, 30 min), ab255448 cell lysate at 20 µg

Lane 8:

Wild-type HeLa Treated TGF-beta (10 ng/mL, 30 min), ab255448 cell lysate at 20 µg

Lane 9:

SMAD3 knockout HeLa Vehicle Control TGF-beta (0 ng/mL, 30 min), <a href='/en-us/products/cell-lines/human-smad3-knockout-hela-cell-line-ab255431'>ab255431</a> cell lysate at 20 µg

Lane 10:

SMAD3 knockout HeLa Treated TGF-beta (10 ng/mL, 30 min), <a href='/en-us/products/cell-lines/human-smad3-knockout-hela-cell-line-ab255431'>ab255431</a> cell lysate at 20 µg

Lane 11:

Wild-type HEK293 Vehicle Control TGF-beta (0 ng/mL, 30 min), ab259776 cell lysate at 20 µg

Lane 12:

Wild-type HEK293 Treated TGF-beta (10 ng/mL, 30 min), ab259776 cell lysate at 20 µg

Lane 13:

SMAD5 knockout HEK293 Vehicle Control TGF-beta (0 ng/mL, 30 min), <a href='/en-us/products/cell-lines/human-smad5-knockout-hek-293-cell-line-ab269470'>ab269470</a> cell lysate at 20 µg

Lane 14:

SMAD5 knockout HEK293 Treated TGF-beta (10 ng/mL, 30 min), <a href='/en-us/products/cell-lines/human-smad5-knockout-hek-293-cell-line-ab269470'>ab269470</a> cell lysate at 20 µg

Secondary

Lanes 1 - 14:

Goat anti-Rabbit IgG H&L 800CW at 1/20000 dilution

Lanes 1 - 14:

Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution

Observed band size: 52 kDa

false

• WB

Lab

Western blot - Anti-SMAD5 antibody [EP619Y] (AB40771)

Blocking and dilution buffer : 5% NFDM/TBST

All lanes:

Western blot - Anti-SMAD5 antibody [EP619Y] (ab40771) at 1/1000 dilution

Lane 1:

3T3-L1 (Mouse embryonic fibroblast) lysate at 20 µg

Lane 2:

Neuro-2a (Mouse neuroblastoma neuroblast) at 20 µg

Lane 3:

F9 (Mouse embryonal carcinoma epithelial cell) lysate at 20 µg

Secondary

All lanes:

Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated at 1/1000 dilution

Predicted band size: 52 kDa

Observed band size: 52 kDa

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Exposure time: 3min

• WB

Unknown

Western blot - Anti-SMAD5 antibody [EP619Y] (AB40771)

All lanes:

Western blot - Anti-SMAD5 antibody [EP619Y] (ab40771) at 1/1000 dilution

All lanes:

Cos-1 cell lysate at 10 µg

Predicted band size: 52 kDa

Observed band size: 52 kDa

false