Revision 4

#46535

Store at -20C

SMAD4 (D3R4N) XP® Rabbit mAb
CST Logo
Orders:

877-616-CELL (2355)

[email protected]

Support:

877-678-TECH (8324)

Web:

3 Trask Lane | Danvers | Massachusetts | 01923 | USA

For Research Use Only. Not for Use in Diagnostic Procedures.

Applications:
W, IP, IHC-Bond, IHC-P, IF-IC, FC-FP, ChIP, ChIP-seq

Reactivity:
H M R Mk

Sensitivity:
Endogenous

MW (kDa):
70

Source/Isotype:
Rabbit IgG

UniProt ID:
#Q13485

Entrez-Gene Id:
4089

Product Usage Information

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.

Application Dilution
Western Blotting 1:1000
Immunoprecipitation 1:200
IHC Leica Bond 1:400 - 1:1600
Immunohistochemistry (Paraffin) 1:200 - 1:800
Immunofluorescence (Immunocytochemistry) 1:400 - 1:1600
Flow Cytometry (Fixed/Permeabilized) 1:400 - 1:1600
Chromatin IP 1:50
Chromatin IP-seq 1:50

Storage

Supplied in 10 mM sodium HEPES (pH 7.5), 150 mM NaCl, 100 µg/ml BSA, 50% glycerol and less than 0.02% sodium azide. Store at –20°C. Do not aliquot the antibody.

For a carrier free (BSA and azide free) version of this product see product #73885.

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. The epitope has been mapped to residues surrounding Ser315.

Background

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 receptor-regulated SMADs (R-SMADs), which include SMAD1, 2, 3, 5, 9; the common-mediator SMAD (co-SMAD), SMAD4; and the antagonistic or inhibitory SMADs (I-SMADs), SMAD6 and 7 (1-5). Activated type I receptors associate with specific R-SMADs and phosphorylate them on a conserved SSXS motif in the carboxy-terminus. Phosphorylated R-SMADS dissociate from the receptor and form a heteromeric complex with SMAD4, initiating translocation of the heteromeric SMAD complex to the nucleus. Once in the nucleus, SMADs recruit a variety of DNA binding proteins that function to regulate transcriptional activity (6-8).

Background References

Species Reactivity

Species reactivity is determined by testing in at least one approved application (e.g., western blot).

Western Blot Buffer

IMPORTANT: For western blots, incubate membrane with diluted primary antibody in 5% w/v nonfat dry milk, 1X TBS, 0.1% Tween® 20 at 4°C with gentle shaking, overnight.

Applications Key

W: Western Blotting IP: Immunoprecipitation IHC-Bond: IHC Leica Bond IF-IC: Immunofluorescence (Immunocytochemistry) FC-FP: Flow Cytometry (Fixed/Permeabilized) ChIP: Chromatin IP

Cross-Reactivity Key

H: Human M: Mouse R: Rat Hm: Hamster Mk: Monkey Vir: Virus Mi: Mink C: Chicken Dm: D. melanogaster X: Xenopus Z: Zebrafish B: Bovine Dg: Dog Pg: Pig Sc: S. cerevisiae Ce: C. elegans Hr: Horse GP: Guinea Pig Rab: Rabbit G: Goat All: All Species Expected

Trademarks and Patents

Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.

Alexa Fluor is a registered trademark of Life Technologies Corporation.

All other trademarks are the property of their respective owners. Visit cellsignal.com/trademarks for more information.

限制使用

除非 CST 的合法授书代表以书面形式书行明确同意，否书以下条款适用于 CST、其关书方或分书商提供的书品。 任何书充本条款或与本条款不同的客书条款和条件，除非书 CST 的合法授书代表以书面形式书独接受， 否书均被拒书，并且无效。

专品专有“专供研究使用”的专专或专似的专专声明， 且未专得美国食品和专品管理局或其他外国或国内专管机专专专任何用途的批准、准专或专可。客专不得将任何专品用于任何专断或治专目的， 或以任何不符合专专声明的方式使用专品。CST 专售或专可的专品提供专作专最专用专的客专，且专用于研专用途。将专品用于专断、专防或治专目的， 或专专售（专独或作专专成）或其他商专目的而专专专品，均需要 CST 的专独专可。客专：(a) 不得专独或与其他材料专合向任何第三方出售、专可、 出借、捐专或以其他方式专专或提供任何专品，或使用专品制造任何商专专品，(b) 不得复制、修改、逆向工程、反专专、 反专专专品或以其他方式专专专专专品的基专专专或技专，或使用专品开专任何与 CST 的专品或服专专争的专品或服专， (c) 不得更改或专除专品上的任何商专、商品名称、徽专、专利或版专声明或专专，(d) 只能根据 CST 的专品专售条款和任何适用文档使用专品 ， (e) 专遵守客专与专品一起使用的任何第三方专品或服专的任何专可、服专条款或专似专专

Orders: 877-616-CELL (2355) • [email protected] • Support: 877-678-TECH (8324) • [email protected] • Web: cellsignal.com
For Research Use Only. Not for Use in Diagnostic Procedures.

Revision 4

#46535

SMAD4 (D3R4N) XP® Rabbit mAb

CST Logo
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.
Western Blotting Image 1: SMAD4 (D3R4N) XP<sup>®</sup> Rabbit mAb
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.
Immunoprecipitation Image 1: SMAD4 (D3R4N) XP<sup>®</sup> Rabbit mAb
Immunohistochemical analysis of paraffin-embedded human colon carcinoma using SMAD4 (D3R4N) XP® Rabbit mAb performed on the Leica® BOND™ Rx.
Immunohistochemistry Image 1: SMAD4 (D3R4N) XP<sup>®</sup> Rabbit mAb
Orders: 877-616-CELL (2355) • [email protected] • Support: 877-678-TECH (8324) • [email protected] • Web: cellsignal.com
For Research Use Only. Not for Use in Diagnostic Procedures.

Revision 4

#46535

SMAD4 (D3R4N) XP® Rabbit mAb

CST Logo
Immunohistochemical analysis of paraffin-embedded human infiltrating papillary carcinoma of the breast using SMAD4 (D3R4N) XP® Rabbit mAb.
Immunohistochemistry Image 1: SMAD4 (D3R4N) XP<sup>®</sup> Rabbit mAb
Immunohistochemical analysis of paraffin-embedded human ductal carcinoma of the breast using SMAD4 (D3R4N) XP® Rabbit mAb.
Immunohistochemistry Image 2: SMAD4 (D3R4N) XP<sup>®</sup> 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.
Immunohistochemistry Image 3: SMAD4 (D3R4N) XP<sup>®</sup> Rabbit mAb
Orders: 877-616-CELL (2355) • [email protected] • Support: 877-678-TECH (8324) • [email protected] • Web: cellsignal.com
For Research Use Only. Not for Use in Diagnostic Procedures.

Revision 4

#46535

SMAD4 (D3R4N) XP® Rabbit mAb

CST Logo
Immunohistochemical analysis of paraffin-embedded human prostate carcinoma using SMAD4 (D3R4N) XP® Rabbit mAb.
Immunohistochemistry Image 4: SMAD4 (D3R4N) XP<sup>®</sup> 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).
Immunofluorescence Image 1: SMAD4 (D3R4N) XP<sup>®</sup> Rabbit mAb
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.
Flow Cytometry Image 1: SMAD4 (D3R4N) XP<sup>®</sup> Rabbit mAb
Orders: 877-616-CELL (2355) • [email protected] • Support: 877-678-TECH (8324) • [email protected] • Web: cellsignal.com
For Research Use Only. Not for Use in Diagnostic Procedures.

Revision 4

#46535

SMAD4 (D3R4N) XP® Rabbit mAb

CST Logo
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 using SimpleChIP® ChIP-seq DNA Library Prep Kit for Illumina® #56795. The figure shows binding across ID1, a known target gene of Smad4 (see additional figure containing ChIP-qPCR data).
Chromatin Immunoprecipitation Image 1: SMAD4 (D3R4N) XP<sup>®</sup> Rabbit mAb
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 using SimpleChIP® ChIP-seq DNA Library Prep Kit for Illumina® #56795. The figure shows binding across chromosome 20 (upper), including ID1 (lower), a known target gene of Smad4 (see additional figure containing ChIP-qPCR data).
Chromatin Immunoprecipitation Image 2: SMAD4 (D3R4N) XP<sup>®</sup> Rabbit mAb
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.
Chromatin Immunoprecipitation Image 3: SMAD4 (D3R4N) XP<sup>®</sup> Rabbit mAb
Orders: 877-616-CELL (2355) • [email protected] • Support: 877-678-TECH (8324) • [email protected] • Web: cellsignal.com
For Research Use Only. Not for Use in Diagnostic Procedures.