WB
Western blot analysis of Phospho-Smad5 (S463 + S465) on different lysates with Rabbit anti-Phospho-Smad5 (S463 + S465) antibody at 1/1,000 dilution. Lane 1: HeLa whole cell lysate, Lane 2: HeLa treated with 20 ng/mL TGFβ1 for 15 minutes whole cell lysate, Lysates/proteins at 20 µg/Lane. Exposure time: 2 minutes; 4-20% SDS-PAGE gel. Proteins were transferred to a PVDF membrane and blocked with 5% NFDM/TBST for 1 hour at room temperature. The primary antibody at 1/1,000 dilution was used in 5% NFDM/TBST at room temperature for 2 hours. Goat Anti-Rabbit IgG - HRP Secondary Antibody at 1:100,000 dilution was used for 1 hour at room temperature.WB
Western blot analysis of Phospho-Smad5 (S463 + S465) on different lysates with Rabbit anti-Phospho-Smad5 (S463 + S465) antibody at 1/500 dilution. Lane 1: Rat brain tissue lysate, Lane 2: Mouse brain tissue lysate, Lysates/proteins at 20 µg/Lane. Exposure time: 2 minutes; 8% SDS-PAGE gel. Proteins were transferred to a PVDF membrane and blocked with 5% NFDM/TBST for 1 hour at room temperature. The primary antibody at 1/500 dilution was used in 5% NFDM/TBST at room temperature for 2 hours. Goat Anti-Rabbit IgG - HRP Secondary Antibody at 1:300,000 dilution was used for 1 hour at room temperature.IHC
Immunohistochemical analysis of paraffin-embedded human tonsil tissue using anti-Phospho-Smad5 (S463 + S465) antibody. The section was pre-treated using heat mediated antigen retrieval with Tris-EDTA buffer (pH 8.0-8.4) for 20 minutes.The tissues were blocked in 5% BSA for 30 minutes at room temperature, washed with ddH2O and PBS, and then probed with the primary antibody (1/50) for 30 minutes at room temperature. The detection was performed using an HRP conjugated compact polymer system. DAB was used as the chromogen. Tissues were counterstained with hematoxylin and mounted with DPX.IHC
Immunohistochemical analysis of paraffin-embedded mouse brain tissue using anti-Phospho-Smad5 (S463 + S465) antibody. The section was pre-treated using heat mediated antigen retrieval with Tris-EDTA buffer (pH 8.0-8.4) for 20 minutes.The tissues were blocked in 5% BSA for 30 minutes at room temperature, washed with ddH2O and PBS, and then probed with the primary antibody (1/50) for 30 minutes at room temperature. The detection was performed using an HRP conjugated compact polymer system. DAB was used as the chromogen. Tissues were counterstained with hematoxylin and mounted with DPX.IHC
Immunohistochemical analysis of paraffin-embedded rat brain tissue with Rabbit anti-Phospho-Smad5 (S463 + S465) antibody at 1/5,000 dilution. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH 6.0) for 2 minutes. The tissues were blocked in 1% BSA for 20 minutes at room temperature, washed with ddH2O and PBS, and then probed with the primary antibody at 1/5,000 dilution for 1 hour at room temperature. The detection was performed using an HRP conjugated compact polymer system. DAB was used as the chromogen. Tissues were counterstained with hematoxylin and mounted with DPX.FC
Flow cytometric analysis of HeLa+TGFβ1(20ng/mL 15min)(+) cells labeling Phospho-Smad5 (S463 + S465). Cells were fixed and permeabilized. Then stained with the primary antibody (1μg/mL) (red) compared with Rabbit IgG Isotype Control (green). After incubation of the primary antibody at +4℃ for an hour, the cells were stained with a iFluor™ 488 conjugate-Goat anti-Rabbit IgG Secondary antibody at 1/1,000 dilution for 30 minutes at +4℃. Unlabelled sample was used as a control (cells without incubation with primary antibody; black).| Product Name | Phospho-Smad5 (S463 + S465) Recombinant Rabbit Monoclonal Antibody |
|---|---|
| Antibody Type | Primary Antibodies |
| Product description | Smad proteins, the mammalian homologs of the Drosophila Mothers against dpp (Mad) have been implicated as downstream effectors of TGFβ/BMP signaling. Smad1 (also designated Madr1 or JV4-1), Smad5 and mammalian Smad8 (also designated Smad9 or MADH6) are effectors of BMP2 and BMP4 function while Smad2 (also designated Madr2 or JV18-1) and Smad3 are involved in TGFβ and activin-mediated growth modulation. Smad4 (also designated DPC4) has been shown to mediate all of the above activities through interaction with various Smad family members. Smad6 and Smad7 regulate the response to activin/TGFβ signaling by interfering with TGFβ-mediated phosphorylation of other Smad family members. |
| Immunogen | Synthetic phospho-peptide corresponding to residues surrounding Ser463 and 465 of Human Smad5. The immunogen may cross with p-smad1/9. |
| Modification | p-S463/465 |
| Clonality | Monoclonal |
|---|---|
| Isotype | IgG |
| Host Species | Recombinant rabbit |
| Tested Applications | FCICC/IFIHCWB |
| WB:1:1000-1:2000 IHC:1:50-1:200 ICC/IF:1:50-1:200 FC:1:1000 |
|
| Species Reactivity | HumanMouseRat |
| Concentration | 1mg/ml |
| Purification | Protein A |
| Alternative Names | DKFZp781C1895 antibody
DKFZp781O1323 antibody
Dwfc antibody
hSmad5 antibody
JV5 1 antibody
JV5-1 antibody
MAD homolog 5 antibody
MAD mothers against decapentaplegic homolog 5 antibody MADH 5 antibody MADH5 antibody Mothers against decapentaplegic homolog 5 antibody mothers against decapentaplegic drosophila homolog of 5 antibody Mothers against DPP homolog 5 antibody MusMLP antibody SMA and MAD related protein 5 antibody SMAD 5 antibody SMAD family member 5 antibody SMAD mothers against DPP homolog 5 antibody Smad5 antibody SMAD5_HUMAN antibody |
|---|---|
| Molecular Weight(MW) | 52kDa |
| Cellular Localization | Cytoplasm, Nucleus. |
WB
Western blot analysis of Phospho-Smad5 (S463 + S465) on different lysates with Rabbit anti-Phospho-Smad5 (S463 + S465) antibody at 1/1,000 dilution. Lane 1: HeLa whole cell lysate, Lane 2: HeLa treated with 20 ng/mL TGFβ1 for 15 minutes whole cell lysate, Lysates/proteins at 20 µg/Lane. Exposure time: 2 minutes; 4-20% SDS-PAGE gel. Proteins were transferred to a PVDF membrane and blocked with 5% NFDM/TBST for 1 hour at room temperature. The primary antibody at 1/1,000 dilution was used in 5% NFDM/TBST at room temperature for 2 hours. Goat Anti-Rabbit IgG - HRP Secondary Antibody at 1:100,000 dilution was used for 1 hour at room temperature.
WB
Western blot analysis of Phospho-Smad5 (S463 + S465) on different lysates with Rabbit anti-Phospho-Smad5 (S463 + S465) antibody at 1/500 dilution. Lane 1: Rat brain tissue lysate, Lane 2: Mouse brain tissue lysate, Lysates/proteins at 20 µg/Lane. Exposure time: 2 minutes; 8% SDS-PAGE gel. Proteins were transferred to a PVDF membrane and blocked with 5% NFDM/TBST for 1 hour at room temperature. The primary antibody at 1/500 dilution was used in 5% NFDM/TBST at room temperature for 2 hours. Goat Anti-Rabbit IgG - HRP Secondary Antibody at 1:300,000 dilution was used for 1 hour at room temperature.
IHC
Immunohistochemical analysis of paraffin-embedded human tonsil tissue using anti-Phospho-Smad5 (S463 + S465) antibody. The section was pre-treated using heat mediated antigen retrieval with Tris-EDTA buffer (pH 8.0-8.4) for 20 minutes.The tissues were blocked in 5% BSA for 30 minutes at room temperature, washed with ddH2O and PBS, and then probed with the primary antibody (1/50) for 30 minutes at room temperature. The detection was performed using an HRP conjugated compact polymer system. DAB was used as the chromogen. Tissues were counterstained with hematoxylin and mounted with DPX.
IHC
Immunohistochemical analysis of paraffin-embedded mouse brain tissue using anti-Phospho-Smad5 (S463 + S465) antibody. The section was pre-treated using heat mediated antigen retrieval with Tris-EDTA buffer (pH 8.0-8.4) for 20 minutes.The tissues were blocked in 5% BSA for 30 minutes at room temperature, washed with ddH2O and PBS, and then probed with the primary antibody (1/50) for 30 minutes at room temperature. The detection was performed using an HRP conjugated compact polymer system. DAB was used as the chromogen. Tissues were counterstained with hematoxylin and mounted with DPX.
IHC
Immunohistochemical analysis of paraffin-embedded rat brain tissue with Rabbit anti-Phospho-Smad5 (S463 + S465) antibody at 1/5,000 dilution. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH 6.0) for 2 minutes. The tissues were blocked in 1% BSA for 20 minutes at room temperature, washed with ddH2O and PBS, and then probed with the primary antibody at 1/5,000 dilution for 1 hour at room temperature. The detection was performed using an HRP conjugated compact polymer system. DAB was used as the chromogen. Tissues were counterstained with hematoxylin and mounted with DPX.
FC
Flow cytometric analysis of HeLa+TGFβ1(20ng/mL 15min)(+) cells labeling Phospho-Smad5 (S463 + S465). Cells were fixed and permeabilized. Then stained with the primary antibody (1μg/mL) (red) compared with Rabbit IgG Isotype Control (green). After incubation of the primary antibody at +4℃ for an hour, the cells were stained with a iFluor™ 488 conjugate-Goat anti-Rabbit IgG Secondary antibody at 1/1,000 dilution for 30 minutes at +4℃. Unlabelled sample was used as a control (cells without incubation with primary antibody; black).| Application Notes | WB:1:1000-1:2000 IHC:1:50-1:200 ICC/IF:1:50-1:200 FC:1:1000 |
|---|
| Form | Liquid |
|---|---|
| Storage Instructions | Store at +4℃ after thawing. Aliquot store at -20℃ or -80℃. Avoid repeated freeze / thaw cycles. |
| Storage Buffer | 1*TBS (pH7.4), 1%BSA, 40%Glycerol. Preservative: 0.05% Sodium Azide. |
