Smad3 Recombinant Rabbit Monoclonal Antibody [SY25-01]
cat.: ET1607-41
Product Type: Recombinant Rabbit monoclonal IgG, primary antibodies
Species reactivity: Human, Mouse, Rat
Applications: WB, IF-Cell, IF-Tissue, IHC-P, FC
Clonality: Monoclonal
Clone number: SY25-01
Form: Liquid
Storage condition: Store at +4℃ after thawing. Aliquot store at -20℃ or -80℃. Avoid repeated freeze / thaw cycles.
Storage buffer: 1*TBS (pH7.4), 0.05% BSA, 40% Glycerol. Preservative: 0.05% Sodium Azide.
Concentration: 1ug/ul
Purification: Protein A affinity purified.
Molecular weight: Predicted band size: 48 kDa
Isotype: IgG
Immunogen: Synthetic peptide within Human Smad3 aa 181-230 / 425.
Positive control: HeLa cell lysate, A549 cell lysate, HL-60 cell lysate, NIH/3T3 cell lysate, bEnd.3 cell lysate, C6 cell lysate, Wild-type HaCaT whole cell lysate, A549, human lung carcinoma tissue, human breast carcinoma tissue, mouse liver tissue, mouse brain tissue, rat testis tissue, HeLa.
Subcellular location: Cytoplasm, Nucleus.
Recommended Dilutions:
  WB
  IF-Cell
  IF-Tissue
  IHC-P
  FC
1:5,000-1:20,000
1:500
1:50-1:200
1:50-1:200
1:1,000
Uniprot #: SwissProt: P84022 Human | Q8BUN5 Mouse | P84025 Rat
Alternative names: DKFZP586N0721 DKFZp686J10186 hMAD 3 hMAD-3 hSMAD3 HSPC193 HST17436 JV15 2 JV15-2 JV152 LDS1C LDS3 MAD (mothers against decapentaplegic Drosophila) homolog 3 MAD homolog 3 Mad homolog JV15 2 Mad protein homolog MAD, mothers against decapentaplegic homolog 3 Mad3 MADH 3 MADH3 MGC60396 Mothers against decapentaplegic homolog 3 Mothers against DPP homolog 3 SMA and MAD related protein 3 SMAD 3 SMAD SMAD family member 3 SMAD, mothers against DPP homolog 3 Smad3 SMAD3_HUMAN
Images
ET1607-41_1.jpg Fig1: Western blot analysis of Smad3 on different lysates with Rabbit anti-Smad3 antibody (ET1607-41) at 1/20,000 dilution.

Lane 1: HeLa cell lysate
Lane 2: A549 cell lysate
Lane 3: HL-60 cell lysate
Lane 4: NIH/3T3 cell lysate
Lane 5: bEnd.3 cell lysate
Lane 6: C6 cell lysate

Lysates/proteins at 15 µg/Lane.

Predicted band size: 48 kDa
Observed band size: 55 kDa

Exposure time: 1 minute 52 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 (ET1607-41) at 1/20,000 dilution was used in 5% NFDM/TBST at 4℃ overnight. Goat Anti-Rabbit IgG - HRP Secondary Antibody (HA1001) at 1/50,000 dilution was used for 1 hour at room temperature.
ET1607-41_2.jpg Fig2: All lanes: Western blot analysis of Smad3 with anti-Smad3 antibody (ET1607-41) at 1:1,000 dilution.
Lane 1: Wild-type HaCaT whole cell lysate (15 µg).
Lane 2: Smad3 knockout HaCaT whole cell lysate (15 µg).

ET1607-41 was shown to specifically react with Smad3 in wild-type HaCaT cells. NO band was observed when Smad3 knockout sample was tested. Wild-type and Smad3 knockout samples were subjected to SDS-PAGE. Proteins were transferred to a PVDF membrane and blocked with 5% NFDM in TBST for 1 hour at room temperature. The primary antibody (ET1607-41, 1:1,000) was used in 5% BSA at room temperature for 2 hours. Goat anti-Rabbit IgG-HRP antibody at 1:10,000 dilution was used for 1 hour at room temperature.
ET1607-41_3.jpg Fig3: Immunocytochemistry analysis of A549 cells labeling Smad3 with Rabbit anti-Smad3 antibody (ET1607-41) 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-Smad3 antibody (ET1607-41) at 1/500 dilution in 1% BSA in PBST overnight at 4 ℃. Goat Anti-Rabbit IgG H&L (iFluor™ 488, HA1121) 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 (M1305-2, red) was stained at 1/100 dilution overnight at +4℃. Goat Anti-Mouse IgG H&L (iFluor™ 594, HA1126) was used as the secondary antibody at 1/1,000 dilution.
ET1607-41_4.jpg Fig4: Immunohistochemical analysis of paraffin-embedded human lung carcinoma tissue using anti-Smad3 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 (ET1607-41, 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.
ET1607-41_5.jpg Fig5: Immunohistochemical analysis of paraffin-embedded human breast carcinoma tissue using anti-Smad3 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 (ET1607-41, 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.
ET1607-41_6.jpg Fig6: Immunohistochemical analysis of paraffin-embedded mouse liver tissue using anti-Smad3 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 (ET1607-41, 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.
ET1607-41_7.jpg Fig7: Immunohistochemical analysis of paraffin-embedded mouse brain tissue using anti-Smad3 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 (ET1607-41, 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.
ET1607-41_8.jpg Fig8: Immunohistochemical analysis of paraffin-embedded rat testis tissue using anti-Smad3 antibody. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH 6.0) 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 (ET1607-41, 1/200) 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.
ET1607-41_9.jpg Fig9: Immunohistochemical analysis of paraffin-embedded human kidney tissue with Rabbit anti-Smad3 antibody (ET1607-41) at 1/500 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 (ET1607-41) at 1/500 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.
ET1607-41_10.jpg Fig10: Flow cytometric analysis of HeLa cells labeling Smad3.

Cells were fixed and permeabilized. Then stained with the primary antibody (ET1607-41, 1/1,000) (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 (HA1121) at 1/1,000 dilution for 30 minutes at +4℃. Unlabelled sample was used as a control (cells without incubation with primary antibody; black).
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