SMAD4 Recombinant Rabbit mAb (SDT-168-62)

Catalog Number: S0B2218 Application: WB, IHC-P, ICC, IP, ICFCM, ChIP Reactivity: Hu Conjugation: Unconjugated Brand: Starter

Price:

$100 USD

Size: 25μl 100μl 500μl

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Product Details

Product Specification

Host	Rabbit
Antigen	SMAD4
Synonyms	Mothers against decapentaplegic homolog 4; MAD homolog 4; Mothers against DPP homolog 4; Deletion target in pancreatic carcinoma 4; Smad4; hSMAD4
Location	Cytoplasm, Nucleus
Accession	Q13485
Clone Number	SDT-168-62
Antibody Type	Recombinant mAb
Application	WB, IHC-P, ICC, IP, ICFCM, ChIP
Reactivity	Hu
Predicted Reactivity	Bv, Pg, Rt, Ms
Purification	Protein A
Concentration	0.5 mg/ml
Conjugation	Unconjugated
Physical Appearance	Liquid
Storage Buffer	PBS, 40% Glycerol, 0.05% BSA, 0.03% Proclin 300
Stability & Storage	12 months from date of receipt / reconstitution, -20 °C as supplied

Dilution

application	dilution	species
WB	1:1000
IP	1:50
IHC	1:100
ICC	1:100
ICFCM	1:500
ChIP	1:20

Background

SMAD (mothers against decapentaplegic homologs) molecules are the core components in TGF-β signaling pathway. TGF-β binding to its receptor induces phosphorylation and activation of receptor-regulated SMADs (R-SMADs), SMAD2 and SMAD3, which subsequently associate with their partner SMAD4 and translocate from cytoplasm to nucleus. Formation of R-SMAD–SMAD4 complexes is essential in signaling of most TGF-β family members.

Picture

Western Blot

WB result of SMAD4 Rabbit mAb
Primary antibody: SMAD4 Rabbit mAb at 1/1000 dilution
Lane 1: HT-29 whole cell lysate 20 µg
Lane 2: HepG2 whole cell lysate 20 µg
Lane 3: HCT 116 whole cell lysate 20 µg
Lane 4: Jurkat whole cell lysate 20 µg
Negative control: HT-29 whole cell lysate
Secondary antibody: Goat Anti-Rabbit IgG, (H+L), HRP conjugated at 1/10000 dilution
Predicted MW: 60 kDa
Observed MW: 65 kDa

FC

Flow cytometric analysis of HT-29 (Human colorectal adenocarcinoma epithelial cell, left) / HepG2 (Human hepatocellular carcinoma epithelial cell, right) cells labelling SMAD4 antibody at 1/500 dilution (0.1 μg)/ (red) compared with a Rabbit monoclonal IgG (Black) isotype control and an unlabelled control (cells without incubation with primary antibody and secondary antibody) (Blue). Goat Anti-Rabbit IgG Alexa Fluor® 488 was used as the secondary antibody. Negative control: HT-29 cells

IP

SMAD4 Recombinant Rabbit mAb at 1/50 dilution (1 µg) immunoprecipitating SMAD4 in 0.4 mg HeLa whole cell lysate.
Western blot was performed on the immunoprecipitate using SMAD4 Recombinant Rabbit mAb at 1/1000 dilution.
Secondary antibody (HRP) for IP was used at 1/1000 dilution.
Lane 1: HeLa whole cell lysate 40 µg (Input)
Lane 2: SMAD4 Recombinant Rabbit mAb IP in HeLa whole cell lysate
Lane 3: Rabbit monoclonal IgG IP in HeLa whole cell lysate
Predicted MW: 60 kDa
Observed MW: 65 kDa

Immunohistochemistry

IHC shows positive staining in paraffin-embedded human colon cancer. Anti-SMAD4 antibody was used at 1/100 dilution, followed by a HRP Polymer for Mouse & Rabbit IgG (ready to use). Counterstained with hematoxylin. Heat mediated antigen retrieval with Tris/EDTA buffer pH9.0 was performed before commencing with IHC staining protocol.

IHC shows positive staining in paraffin-embedded human lung squamous cell carcinoma. Anti-SMAD4 antibody was used at 1/100 dilution, followed by a HRP Polymer for Mouse & Rabbit IgG (ready to use). Counterstained with hematoxylin. Heat mediated antigen retrieval with Tris/EDTA buffer pH9.0 was performed before commencing with IHC staining protocol.

IHC shows positive staining in paraffin-embedded human pancreatic cancer (Loss of SMAD4 expression in tumors but nuclear expression in stromal cells). Anti-SMAD4 antibody was used at 1/100 dilution, followed by a HRP Polymer for Mouse & Rabbit IgG (ready to use). Counterstained with hematoxylin. Heat mediated antigen retrieval with Tris/EDTA buffer pH9.0 was performed before commencing with IHC staining protocol.

IHC shows positive staining in paraffin-embedded human pancreatic cancer (Loss of SMAD4 expression in tumors but expression in stromal and paracancerous cells). Anti-SMAD4 antibody was used at 1/100 dilution, followed by a HRP Polymer for Mouse & Rabbit IgG (ready to use). Counterstained with hematoxylin. Heat mediated antigen retrieval with Tris/EDTA buffer pH9.0 was performed before commencing with IHC staining protocol.

Immunocytochemistry

ICC shows positive staining in HepG2 cells. Anti-SMAD4 antibody was used at 1/100 dilution (Green) and incubated overnight at 4°C. Goat polyclonal Antibody to Rabbit IgG - H&L (Alexa Fluor® 488) was used as secondary antibody at 1/1000 dilution. The cells were fixed with 4% PFA and permeabilized with 0.1% PBS-Triton X-100. Nuclei were counterstained with DAPI (Blue).

Negative control: ICC shows negative staining in HT-29 cells. Anti-SMAD4 antibody was used at 1/100 dilution and incubated overnight at 4°C. Goat polyclonal Antibody to Rabbit IgG - H&L (Alexa Fluor® 488) was used as secondary antibody at 1/1000 dilution. The cells were fixed with 4% PFA and permeabilized with 0.1% PBS-Triton X-100. Nuclei were counterstained with DAPI (Blue).

ChIP

Chromatin immunoprecipitation (ChIP) was performed on HaCaT+TGF-β(7ng/mL,1h) cells cross - linked with 1% formaldehyde for 10 min, then chromatin was fragmented by sonication. Parallel reactions used SMAD4 Recombinant Rabbit mAb (SDT-168-62) and Rabbit mAb IgG Isotype Control (SDT-R173) at 1:20 for immunoprecipitation.
Post - immunoprecipitation, both samples were washed, eluted, and cross - links reversed. Purified DNA was analyzed by qPCR.
qPCR showed the enrichment of ID1, CDKN1A and SAT-α in
SMAD4 Recombinant Rabbit mAb (SDT-168-62)
-immunoprecipitated sample.