Smad5 Antibody (YA073)

Smad5 Antibody (YA073) is a Rabbit-derived and non-conjugated IgG monoclonal antibody, targeting to Smad5.

Smad1/5/9 is a transcriptional regulator involved in various cellular processes such as embryonic development, cell differentiation, angiogenesis, and tissue homeostasis. Upon BMP ligand binding to cell surface receptors, it is phosphorylated by activated type I BMP receptors (BMPRIs) and associates with SMAD4 to form a heteromeric complex that translocates into the nucleus, functioning as a transcription factor. The hetero-trimeric complex recognizes cis-regulatory elements containing Smad Binding Elements (SBEs) to modulate signaling network outcomes. Non-phosphorylated SMAD5 plays a cytoplasmic role in energy metabolism regulation by promoting mitochondrial respiration and glycolysis in response to cytoplasmic pH changes. Mechanistically, it interacts with hexokinase 1 (HK1) to accelerate glycolysis.

Free

Q99717

Predicted band size: 52 kDa; Observed band size: 60 kDa

Protein A affinity purified.

Cytoplasm, Nucleus.

Non-conjugated

Unmodified

AB_3102889

Signal Transduction

Primary Antibody; Recombinant Rabbit Monoclonal Antibody

Recombinant, Monoclonal

Rabbit

Human, Mouse

WB: 1:1,000-1:2,000 ;ICC: 1:50-1:200 ;IHC-P: 1:50-1:200 ;FC: 1:50-1:100

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  Western blot analysis of extracts from 3T3-L1(lane2(20μg) and 3T3-L1(lane 3(40μg) ,HEK293(lane 4(20ug)and HEK293(lane 5(40μg) using Smad5 Antibody (HY-P80327) Rabbit mAb. Proteins were transferred to a PVDF membrane and blocked with 5% non-fat milk in TBST for 2 hour at room temperature. The primary antibody (1/1000) and Loading control antibody (Beta Actin, HY-P80438, 1/10000) was used in 5% non-fat milk in TBST at 4°C overnight. Goat Anti-Mouse/Rabbit IgG-HRP Secondary Antibody (1/10000) was used for 1 hour at room temperature.
• 
  Immunocytochemistry analysis of Hela cells labeling Smad5 with Smad5 Antibody (HY-P80327) at 1/50 dilution. Cells were fixed in 4% paraformaldehyde for 15 minutes at room temperature, permeabilized with 0.1% Triton X-100 for 10 minutes at room temperature, then blocked with QuickBlock™ Blocking Buffer for Immunol Staining for 10 min at room temperature. Cells were then incubated with Smad5 Antibody (HY-P80327) at 1/50 dilution in QuickBlock™ Blocking Buffer for Immunol Staining at 4 ℃. Alexa Fluor® 488-conjugated AffiniPure Goat Anti-Rabbit IgG H&L（HY-P8002, Green) 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. The Nuclear counterstain was DAPI (Blue).
• 
  Immunocytochemistry analysis of A549 cells labeling Smad5 with Smad5 Antibody (HY-P80327) at 1/50 dilution. Cells were fixed in 4% paraformaldehyde for 15 minutes at room temperature, permeabilized with 0.1% Triton X-100 for 10 minutes at room temperature, then blocked with QuickBlock™ Blocking Buffer for Immunol Staining for 10 min at room temperature. Cells were then incubated with Smad5 Antibody (HY-P80327)at 1/50 dilution in QuickBlock™ Blocking Buffer for Immunol Staining at 4 ℃. Alexa Fluor® 488-conjugated AffiniPure Goat Anti-Rabbit IgG H&L（HY-P8002,Green) 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. The Nuclear counterstain was DAPI (Blue).
• 
  Immunohistochemical analysis of paraffin-embedded rat colon tissue using Rb Antibody. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH 6.0) for 8 minutes. The tissues were blocked in QuickBlock for 20 minutes at room temperature, washed with ddH2O and PBS, and then probed with the primary antibody (HY-P80300, 1:500) in 4℃ overnight. 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.
• 
  Immunohistochemical analysis of paraffin-embedded rat colon tissue using Rb Antibody. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH 6.0) for 8 minutes. The tissues were blocked in QuickBlock for 20 minutes at room temperature, washed with ddH2O and PBS, and then probed with the primary antibody (HY-P80300, 1:500) in 4℃ overnight. 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.
• 
  Flow cytometric analysis of 1X10^6 Hela cells labeling Smad5 Antibody(HY-P80327, red). Cells were fixed with 4% paraformaldehyde and permeabilised with 90% methanol. Then stained with the primary antibody at 1/50 dilution for an hour at 4℃. Alexa Fluor® 488-conjugated AffiniPure Goat Anti-Rabbit IgG H&L (HY-P8002) was used as the secondary antibody at 1/1,000 dilution for 30 minutes at 4℃. Rabbit IgG Isotype Control (HY-P80879, blue) was used as the isotype control, cells without incubation with primary antibody were used as the unlabeled control (black).

WB, ICC/IF, IHC-P, FC

Liquid

Supplied in 1*TBS (pH7.4), 0.05% BSA and 40% Glycerol. Preservative: 0.05% Sodium Azide.

Stored at -20°C for 1 year. Avoid repeated freeze / thaw cycles.

Shipping with blue ice.

IgG

Endogenous

Synthetic peptide corresponding to Human Smad5.AA range:231-282.