Revision 7

#8685

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Orders:

877-616-CELL (2355)

[email protected]

Support:

877-678-TECH (8324)

3 Trask Lane | Danvers | Massachusetts | 01923 | USA

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

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

Reactivity:
H M R Mk

Sensitivity:
Endogenous

MW (kDa):
52, 60

Source/Isotype:
Rabbit IgG

UniProt ID:
#P84022, #Q15796

Entrez-Gene Id:
4088, 4087

Product Usage Information

For optimal ChIP and ChIP-seq results, use 5 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 1:100
Immunofluorescence (Immunocytochemistry) 1:400 - 1:800
Flow Cytometry (Fixed/Permeabilized) 1:100 - 1:400
Chromatin IP 1:100
Chromatin IP-seq 1:100

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 #55607.

Specificity/Sensitivity

SMAD2/3 (D7G7) Rabbit Monoclonal Antibody recognizes endogenous levels of total SMAD2/3 protein.

Source / Purification

Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues surrounding His198 of human SMAD2/3 protein.

Background

Members of the SMAD family of signal transduction molecules are components of a critical intracellular pathway that transmit 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, and 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 carboxy-terminal SSXS motif. The 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).

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 BSA, 1X TBS, 0.1% Tween® 20 at 4°C with gentle shaking, overnight.

Applications Key

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

Cross-Reactivity Key

H: Human M: Mouse R: Rat Mk: Monkey

Trademarks and Patents

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

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

Limited Uses

Except as otherwise expressly agreed in a writing signed by a legally authorized representative of CST, the following terms apply to Products provided by CST, its affiliates or its distributors. Any Customer's terms and conditions that are in addition to, or different from, those contained herein, unless separately accepted in writing by a legally authorized representative of CST, are rejected and are of no force or effect.

Products are labeled with For Research Use Only or a similar labeling statement and have not been approved, cleared, or licensed by the FDA or other regulatory foreign or domestic entity, for any purpose. Customer shall not use any Product for any diagnostic or therapeutic purpose, or otherwise in any manner that conflicts with its labeling statement. Products sold or licensed by CST are provided for Customer as the end-user and solely for research and development uses. Any use of Product for diagnostic, prophylactic or therapeutic purposes, or any purchase of Product for resale (alone or as a component) or other commercial purpose, requires a separate license from CST. Customer shall (a) not sell, license, loan, donate or otherwise transfer or make available any Product to any third party, whether alone or in combination with other materials, or use the Products to manufacture any commercial products, (b) not copy, modify, reverse engineer, decompile, disassemble or otherwise attempt to discover the underlying structure or technology of the Products, or use the Products for the purpose of developing any products or services that would compete with CST products or services, (c) not alter or remove from the Products any trademarks, trade names, logos, patent or copyright notices or markings, (d) use the Products solely in accordance with CST Product Terms of Sale and any applicable documentation, and (e) comply with any license, terms of service or similar agreement with respect to any third party products or services used by Customer in connection with the Products.

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 7

Cell Signaling Technology Logo
Western blot analysis of extracts from HeLa and ACHN cells using SMAD2/3 (D7G7) XP® Rabbit mAb.
Western Blotting Image 1: SMAD2/3 (D7G7) Rabbit Monoclonal Antibody
Immunoprecipitation of SMAD2/3 protein from HCT116 extracts. Lane 1 is 10% input, lane 2 is Rabbit (DA1E) mAb IgG XP® Isotype Control #3900, and lane 3 is SMAD2/3 (D7G7) XP®. Western blot analysis was performed using SMAD2/3 (D7G7) XP®. Mouse Anti-rabbit IgG (Conformation Specific) (L27A9) mAb #3678 was used as a secondary antibody.
Immunoprecipitation Image 1: SMAD2/3 (D7G7) Rabbit Monoclonal Antibody
Confocal immunofluorescent analysis of HeLa cells, serum starved (left), treated with hTGF-β3 #8425 (100 ng/ml, 30 min, center), or treated with hTGF-β3 and SB43152 (10 ug/mL, 1 hr, right), using SMAD2/3 (D7G7) XP® Rabbit mAb (green). Actin filaments were labeled with DY-554 phalloidin (red).
Immunofluorescence Image 1: SMAD2/3 (D7G7) Rabbit Monoclonal Antibody
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 7

Cell Signaling Technology Logo
Flow cytometric analysis of HeLa cells using SMAD2/3 (D7G7) XP® Rabbit mAb (solid line) compared to concentration-matched Rabbit (DA1E) mAb IgG XP® Isotype Control #3900 (dashed line). Anti-rabbit IgG (H+L), F(ab')2 Fragment (Alexa Fluor® 488 Conjugate) #4412 was used as a secondary control.
Flow Cytometry Image 1: SMAD2/3 (D7G7) Rabbit Monoclonal Antibody
Chromatin immunoprecipitations were performed with cross-linked chromatin from HaCaT cells treated with hTGF-β3 #8425 (7 ng/ml, 1 hr) and SMAD2/3 (D7G7) XP® Rabbit mAb, using SimpleChIP® Enzymatic Chromatin IP Kit (Magnetic Beads) #9003. DNA Libraries were prepared using DNA Library Prep Kit for Illumina® (ChIP-seq, CUT&RUN) #56795. The figure shows binding across ID1, a known target gene of SMAD2/3 (see additional figure containing ChIP-qPCR data). For additional ChIP-seq tracks, please download the product datasheet.
Chromatin Immunoprecipitation Image 1: SMAD2/3 (D7G7) Rabbit Monoclonal Antibody
Chromatin immunoprecipitations were performed with cross-linked chromatin from HaCaT cells treated with hTGF-β3 #8425 (7 ng/ml, 1 hr) and SMAD2/3 (D7G7) XP® Rabbit mAb, using SimpleChIP® Enzymatic Chromatin IP Kit (Magnetic Beads) #9003. DNA Libraries were prepared using DNA Library Prep Kit for Illumina® (ChIP-seq, CUT&RUN) #56795. The figure shows binding across chromosome 20 (upper), including ID1 (lower), a known target gene of SMAD2/3 (see additional figure containing ChIP-qPCR data).
Chromatin Immunoprecipitation Image 2: SMAD2/3 (D7G7) Rabbit Monoclonal Antibody
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 7

Cell Signaling Technology Logo
Chromatin immunoprecipitations were performed with cross-linked chromatin from HaCaT cells treated with hTGF-β3 #8425 (7 ng/ml, 1 hr) and either SMAD2/3 (D7G7) XP® Rabbit mAb or Normal Rabbit IgG #2729 using SimpleChIP® Enzymatic Chromatin IP Kit (Magnetic Beads) #9003. The enriched DNA was quantified by real-time PCR using SimpleChIP® Human CDKN1A Intron 1 Primers #4669, SimpleChIP® Human ID1 Promoter Primers #5139, 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: SMAD2/3 (D7G7) Rabbit Monoclonal Antibody
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.