SMAD4 (D3R4N) XP® Rabbit mAbProduct information
Product Pathways - TGF-beta/Smad Signaling
SMAD4 (D3R4N) XP® Rabbit mAb #46535
|46535S||100 µl (10 western blots)||---||In Stock||---|
|46535T||20 µl (2 western blots)||---||In Stock||---|
|46535||carrier free and custom formulation / quantity||email request|
|W||1:1000||Human, Mouse, Rat, Monkey||Endogenous||70||Rabbit IgG|
Species cross-reactivity is determined by western blot.
Applications Key: W=Western Blotting, IP=Immunoprecipitation, IHC-P=Immunohistochemistry (Paraffin), IF-IC=Immunofluorescence (Immunocytochemistry), F=Flow Cytometry, ChIP=Chromatin IP, ChIP-seq=Chromatin IP-seq
Directions For Use
For optimal ChIP and ChIP-seq results, use 10 μl of antibody and 10 μg of chromatin (approximately 4 x 106 cells) per IP. This antibody has been validated using SimpleChIP® Enzymatic Chromatin IP Kits.
Specificity / Sensitivity
SMAD4 (D3R4N) XP® Rabbit mAb recognizes endogenous levels of total SMAD4 protein.
Source / Purification
Monoclonal antibody is produced by immunizing animals with recombinant protein corresponding to human SMAD4 protein.
Western blot analysis of extracts from various cell lines using SMAD4 (D3R4N) XP® Rabbit mAb (upper) and β-Actin (D6A8) Rabbit mAb #8457 (lower). Specificity of the antibody is confirmed by the absence of signal in extracts from HT-29 and COLO 205 cells, both of which contain Smad4 null mutations.
Immunoprecipitation of SMAD4 from HCT 116 cell extracts. Lane 1 is 10% input, lane 2 is Rabbit (DA1E) mAb IgG XP® Isotype Control #3900, and lane 3 is SMAD4 (D3R4N) XP® Rabbit mAb. Western blot analysis was performed using SMAD4 (D3R4N) XP® Rabbit mAb. Anti-Rabbit IgG, HRP-linked Antibody #7074 was used as the secondary antibody.
Immunohistochemical analysis of paraffin-embedded human infiltrating papillary carcinoma of the breast using SMAD4 (D3R4N) XP® Rabbit mAb.
Immunohistochemical analysis of paraffin-embedded human ductal carcinoma of the breast using SMAD4 (D3R4N) XP® Rabbit mAb.
Immunohistochemical analysis of paraffin-embedded COLO 320 cell pellet (left, positive) or COLO 205 cell pellet (right, negative) using SMAD4 (D3R4N) XP® Rabbit mAb.
Immunohistochemical analysis of paraffin-embedded human prostate carcinoma using SMAD4 (D3R4N) XP® Rabbit mAb.
Confocal immunofluorescent analysis of HaCaT cells (positive, top panel) or HT-29 cells (negative, bottom panel), either serum-starved (left panel) or serum-starved and treated with Human Transforming Growth Factor β1 (hTGF-β1) #8915 (7 ng/mL, 1 hr; right panel), using Smad4 (D3R4N) XP® Rabbit mAb (green). Actin filaments were labeled wtih DyLight™ 554 Phalloidin #13054 (red).
Flow cytometric analysis of HT-29 cells (blue) and HCT 116 cells (green) using SMAD4 (D3R4N) XP® Rabbit mAb (solid lines) or a concentration matched Rabbit (DA1E) mAb IgG XP® Isotype Control #3900 (dashed lines). Anti-rabbit IgG (H+L), F(ab')2 Fragment (Alexa Fluor® 488 Conjugate) #4412 was used as a secondary antibody.
Chromatin immunoprecipitations were performed with cross-linked chromatin from HaCaT cells treated with TGF-β1 #8915 (7 ng/mL, 1 hr) and either Smad4 (D3R4N) XP® Rabbit mAb (upper) or Smad4 (D3M6U) Rabbit mAb #38454 (lower), using SimpleChIP® Plus Enzymatic Chromatin IP Kit (Magnetic Beads) #9005. DNA Libraries were prepared from 5 ng enriched ChIP DNA using NEBNext® Ultra™ II DNA Library Prep Kit for Illumina®, and sequenced on the Illumina NextSeq. The figure shows binding across ID1, a known target gene of Smad4 (see additional figure containing ChIP-qPCR data). For additional ChIP-seq tracks, please download the product data sheet.
Chromatin immunoprecipitations were performed with cross-linked chromatin from HaCaT cells treated with TGFβ1 #8915 (7 ng/ml, 1h), and either SMAD4 (D3R4N) XP® Rabbit mAb or Normal Rabbit IgG #2729 using SimpleChIP® Plus Enzymatic Chromatin IP Kit (Magnetic Beads) #9005. The enriched DNA was quantified by real-time PCR using SimpleChIP® Human ID1 Promoter Primers #5139, human JunB promoter primers, and SimpleChIP® Human α Satellite Repeat Primers #4486. The amount of immunoprecipitated DNA in each sample is represented as signal relative to the total amount of input chromatin, which is equivalent to one.
Members of the Smad family of signal transduction molecules are components of a critical intracellular pathway that transmits TGF-β signals from the cell surface into the nucleus. Three distinct classes of Smads have been defined: the recepter-regulated Smads (R-Smads), which include Smad1, 2, 3, 5, 8; the common-mediator Smad (co-Smad), Smad4; and the antagonistic or inhibitory Smads (I-Smads), Smad6 and 7 (1-5). Briefly, activated type I receptors associate with specific R-Smads and phosphorylate them on a conserved SSXS motif at the carboxy-terminus of the proteins. The phosphorylated R-Smad dissociates from the receptor and forms a heteromeric complex with the co-Smad, Smad4, and together the complex moves to the nucleus. Once in the nucleus, Smads can target a variety of DNA binding proteins to regulate transcriptional responses (6-8).
- Heldin, C. H. et al. (1997) Nature 390, 465-71.
- Attisano, L. and Wrana, J.L. (1998) Curr. Opin. Cell Biol. 10, 188-94.
- Derynck, R. et al. (1998) Cell 95, 737-40.
- Massague, J. (1998) Annu. Rev. Biochem. 67, 753-91.
- Whitman, . (1998) Genes Dev. 12, 2445-62.
- Wrana, J. (2000) Science 23, 1-9.
- Attisano, L. and Wrana, J. (2002) Science 296, 1646-7.
- Moustakas, A. et al. (2001) J. Cell Sci. 114, 4359-69.
Have you published research involving the use of our products? If so we'd love to hear about it. Please let us know!
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This product is intended for research purposes only. The product is not intended to be used for therapeutic or diagnostic purposes in humans or animals.
Ultra is a trademark of New England Biolabs, Inc.
Illumina is a registered trademark of Illumina, Inc.
Tween is a registered trademark of ICI Americas, Inc.
SignalStain is a trademark of Cell Signaling Technology, Inc.
NEBNext is a registered trademark of New England Biolabs, Inc.
XP is a registered trademark of Cell Signaling Technology, Inc.
SimpleChIP is a registered trademark of Cell Signaling Technology, Inc.
Alexa Fluor is a registered trademark of Life Technologies Corporation.
Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.
DyLight is a trademark of Thermo Fisher Scientific, Inc. and its subsidiaries.