WB
Western blot analysis of Phospho-SMAD2 (S465+467) on different lysates with Rabbit anti-Phospho-SMAD2 (S465+467) antibody at 1/1,000 dilution. Lane 1: HeLa cell lysate, Lane 2: HeLa starved overnight then treated with 10ng/mL TGF-β1 for 30 minutes cell lysate, Lane 3: NIH/3T3 cell lysate, Lane 4: NIH/3T3 starved overnight then treated with 10ng/mL TGF-β1 for 30 minutes cell lysate, Lysates/proteins at 20 µg/Lane. Exposure time: 1 minute 20 seconds; 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 4℃ overnight. Goat Anti-Rabbit IgG - HRP Secondary Antibody at 1/50,000 dilution was used for 1 hour at room temperature.IHC
Immunohistochemical analysis of paraffin-embedded human stomach tissue with Rabbit anti-Phospho-SMAD2 (S465+467) antibody at 1/200 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 1/200 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.ICC/IF
Immunocytochemistry analysis of NIH/3T3 cells treated with or without 20ng/mL TGF-β1 for 15 minutes labeling Phospho-SMAD2 (S465+467) with Rabbit anti-Phospho-SMAD2 (S465+467) antibody at 1/500 dilution. Cells were fixed in 4% paraformaldehyde for 20 minutes at room temperature, permeabilized with 0.1% Triton X-100 in PBS for 5 minutes at room temperature, then blocked with 1% BSA in 10% negative goat serum for 1 hour at room temperature. Cells were then incubated with Rabbit anti-Phospho-SMAD2 (S465+467) antibody at 1/500 dilution in 1% BSA in PBST overnight at 4 ℃. Goat Anti-Rabbit IgG H&L (iFluor™ 488) was used as the secondary antibody at 1/1,000 dilution. PBS instead of the primary antibody was used as the secondary antibody only control. Nuclear DNA was labelled in blue with DAPI. Beta tubulin (red) was stained at 1/100 dilution overnight at +4℃. Goat Anti-Mouse IgG H&L (iFluor™ 594) was used as the secondary antibody at 1/1,000 dilution.| Product Name | Phospho-SMAD2 (S465+467) Recombinant Rabbit Monoclonal Antibody |
|---|---|
| Antibody Type | Primary Antibodies |
| Product description |
|
| Immunogen | Synthetic phospho-peptide corresponding to residues surrounding Ser465 and 467 of Human SMAD2. |
| Modification | p-S465+467 |
| Clonality | monoclonal |
|---|---|
| Isotype | IgG |
| Host Species | Recombinant rabbit |
| Tested Applications | ICC/IFIHCWB |
| WB:1:1000 IHC:1:200 ICC/IF:1:500 |
|
| Species Reactivity | HumanMouseRat |
| Concentration | 1mg/ml |
| Purification | Protein A |
| Alternative Names | Drosophila homolog of MADR2 antibody hMAD-2 antibody HsMAD2 antibody JV18 antibody JV18-1 antibody JV181 antibody MAD antibody MAD homolog 2 antibody MAD Related Protein 2 antibody Mad-related protein 2 antibody MADH2 antibody MADR2 antibody MGC22139 antibody MGC34440 antibody Mother against DPP homolog 2 antibody Mothers against decapentaplegic homolog 2 antibody Mothers against decapentaplegic Drosophila homolog of 2 antibody Mothers against DPP homolog 2 antibody OTTHUMP00000163489 antibody Sma and Mad related protein 2 antibody Sma- and Mad-related protein 2 MAD antibody SMAD 2 antibody SMAD family member 2 antibody SMAD mothers against DPP homolog 2 antibody SMAD2 antibody SMAD2_HUMAN antibody |
|---|---|
| Molecular Weight(MW) | 52kDa(Observed band size: 60kDa) |
| Function | R-SMADs are receptor-regulated SMADs. SMADs are transcription factors that transduce extracellular TGF-β superfamily ligand signaling from cell membrane bound TGF-β receptors into the nucleus where they activate transcription TGF-β target genes. R-SMADS are directly phosphorylated on their c-terminus by type 1 TGF-β receptors through their intracellular kinase domain, leading to R-SMAD activation. R-SMADS include SMAD2 and SMAD3 from the TGF-β/Activin/Nodal branch, and SMAD1, SMAD5 and SMAD8 from the BMP/GDP branch of TGF-β signaling. In response to signals by the TGF-β superfamily of ligands these proteins associate with receptor kinases and are phosphorylated at an SSXS motif at their extreme C-terminus. These proteins then typically bind to the common mediator Smad or co-SMAD SMAD4. |
| Cellular Localization | Cytoplasm, Nucleus. |
WB
Western blot analysis of Phospho-SMAD2 (S465+467) on different lysates with Rabbit anti-Phospho-SMAD2 (S465+467) antibody at 1/1,000 dilution. Lane 1: HeLa cell lysate, Lane 2: HeLa starved overnight then treated with 10ng/mL TGF-β1 for 30 minutes cell lysate, Lane 3: NIH/3T3 cell lysate, Lane 4: NIH/3T3 starved overnight then treated with 10ng/mL TGF-β1 for 30 minutes cell lysate, Lysates/proteins at 20 µg/Lane. Exposure time: 1 minute 20 seconds; 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 4℃ overnight. Goat Anti-Rabbit IgG - HRP Secondary Antibody at 1/50,000 dilution was used for 1 hour at room temperature.
IHC
Immunohistochemical analysis of paraffin-embedded human stomach tissue with Rabbit anti-Phospho-SMAD2 (S465+467) antibody at 1/200 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 1/200 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.
ICC/IF
Immunocytochemistry analysis of NIH/3T3 cells treated with or without 20ng/mL TGF-β1 for 15 minutes labeling Phospho-SMAD2 (S465+467) with Rabbit anti-Phospho-SMAD2 (S465+467) antibody at 1/500 dilution. Cells were fixed in 4% paraformaldehyde for 20 minutes at room temperature, permeabilized with 0.1% Triton X-100 in PBS for 5 minutes at room temperature, then blocked with 1% BSA in 10% negative goat serum for 1 hour at room temperature. Cells were then incubated with Rabbit anti-Phospho-SMAD2 (S465+467) antibody at 1/500 dilution in 1% BSA in PBST overnight at 4 ℃. Goat Anti-Rabbit IgG H&L (iFluor™ 488) was used as the secondary antibody at 1/1,000 dilution. PBS instead of the primary antibody was used as the secondary antibody only control. Nuclear DNA was labelled in blue with DAPI. Beta tubulin (red) was stained at 1/100 dilution overnight at +4℃. Goat Anti-Mouse IgG H&L (iFluor™ 594) was used as the secondary antibody at 1/1,000 dilution.| Application Notes | WB:1:1000 IHC:1:200 ICC/IF:1:500 |
|---|
| 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. |
