Recombinant Anti-Smad2 (phospho S250) antibody [EPR26263-58] KO Tested (ab300079)

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Western blot - Anti-Smad2 (phospho S250) antibody [EPR26263-58] (AB300079)

Key features and details

Produced recombinantly (animal-free) for high batch-to-batch consistency and long term security of supply
Rabbit monoclonal [EPR26263-58] to Smad2 (phospho S250)
Suitable for: WB, Dot blot, IP, ICC/IF
Knockout validated
Reacts with: Mouse, Rat, Human

Product name

Anti-Smad2 (phospho S250) antibody [EPR26263-58]
See all Smad2 primary antibodies
Description

Rabbit monoclonal [EPR26263-58] to Smad2 (phospho S250)
Host species

Rabbit
Tested applications

Suitable for: WB, Dot blot, IP, ICC/IFmore details
Unsuitable for: Flow Cyt (Intra) or IHC-P
Species reactivity

Reacts with: Mouse, Rat, Human
Immunogen

This product was produced with the following immunogens:
Synthetic peptide. This information is proprietary to Abcam and/or its suppliers.

Synthetic peptide. This information is proprietary to Abcam and/or its suppliers.
Positive control
- WB: Whole cell lysates: Parental HeLa (human cervix adenocarcinoma epithelial cell) untreated and treated with 0.2µM PMA for 0.5h, NIH/3T3 untreated and treated with 0.2µM PMA for 0.5h, Untreated and treated with 0.2µM PMA for 0.5h C6 (rat glial tumor glial cell), HeLa treated with 0.2µM PMA for 0.5h (Untreated membrane ). ICC/IF: HeLa IP: HeLa and HIH/3T3 treated with 0.2µM PMA for 0.5h. DB: Smad2 peptides phosphorylated at: S245+S250+S255 a, S245+S250+S255 b, S245+S250, S250+S255, S250 only.
General notes
This product is a recombinant monoclonal antibody, which offers several advantages including:
- - High batch-to-batch consistency and reproducibility
- - Improved sensitivity and specificity
- - Long-term security of supply
- - Animal-free production
For more information see here.
Our RabMAb^® technology is a patented hybridoma-based technology for making rabbit monoclonal antibodies. For details on our patents, please refer to RabMAb^® patents.

Form

Liquid
Storage instructions

Shipped at 4°C. Store at +4°C short term (1-2 weeks). Upon delivery aliquot. Store at -20°C long term. Avoid freeze / thaw cycle.
Storage buffer

pH: 7.20
Preservative: 0.01% Sodium azide
Constituents: 59% PBS, 40% Glycerol (glycerin, glycerine), 0.05% BSA
Concentration information loading...
Purity

Protein A purified
Clonality

Monoclonal
Clone number

EPR26263-58
Isotype

IgG
Research areas

The Abpromise guarantee

Our Abpromise guarantee covers the use of ab300079 in the following tested applications.

The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.

Application	Abreviews	Notes
WB		1/1000. Detects a band of approximately 65 kDa (predicted molecular weight: 52 kDa).
Dot blot		1/1000.
IP		1/30.
ICC/IF		1/50.

Notes
WB 1/1000. Detects a band of approximately 65 kDa (predicted molecular weight: 52 kDa).
Dot blot 1/1000.
IP 1/30.
ICC/IF 1/50.

Application notes

Is unsuitable for Flow Cyt (Intra) or IHC-P.

Function

Receptor-regulated SMAD (R-SMAD) that is an intracellular signal transducer and transcriptional modulator activated by TGF-beta (transforming growth factor) and activin type 1 receptor kinases. Binds the TRE element in the promoter region of many genes that are regulated by TGF-beta and, on formation of the SMAD2/SMAD4 complex, activates transcription. May act as a tumor suppressor in colorectal carcinoma.
Tissue specificity

Expressed at high levels in skeletal muscle, heart and placenta.
Sequence similarities

Belongs to the dwarfin/SMAD family.
Contains 1 MH1 (MAD homology 1) domain.
Contains 1 MH2 (MAD homology 2) domain.
Post-translational
modifications

Phosphorylated on one or several of Thr-220, Ser-245, Ser-250, and Ser-255. In response to TGF-beta, phosphorylated on Ser-465/467 by TGF-beta and activin type 1 receptor kinases. Able to interact with SMURF2 when phosphorylated on Ser-465/467, recruiting other proteins, such as SNON, for degradation. In response to decorin, the naturally occurring inhibitor of TGF-beta signaling, phosphorylated on Ser-240 by CaMK2. Phosphorylated by MAPK3 upon EGF stimulation; which increases transcriptional activity and stability, and is blocked by calmodulin.
In response to TGF-beta, ubiquitinated by NEDD4L; which promotes its degradation.
Acetylated on Lys-19 by coactivators in response to TGF-beta signaling, which increases transcriptional activity. Isoform short: Acetylation increases DNA binding activity in vitro and enhances its association with target promoters in vivo. Acetylation in the nucleus by EP300 is enhanced by TGF-beta.
Cellular localization

Cytoplasm. Nucleus. Cytoplasmic and nuclear in the absence of TGF-beta. On TGF-beta stimulation, migrates to the nucleus when complexed with SMAD4. On dephosphorylation by phosphatase PPM1A, released from the SMAD2/SMAD4 complex, and exported out of the nucleus by interaction with RANBP1.
Target information above from: UniProt accession Q15796 The UniProt Consortium
The Universal Protein Resource (UniProt) in 2010
Nucleic Acids Res. 38:D142-D148 (2010) .

Information by UniProt
Database links
- Entrez Gene: 4087 Human
- Entrez Gene: 17126 Mouse
- Entrez Gene: 29357 Rat
- Omim: 601366 Human
- SwissProt: Q15796 Human
- SwissProt: Q62432 Mouse
- SwissProt: O70436 Rat
- Unigene: 12253 Human
- Unigene: 705764 Human
- Unigene: 391091 Mouse
- Unigene: 2755 Rat
see all
Alternative names
- Drosophila, homolog of, MADR2 antibody
- hMAD-2 antibody
- HsMAD2 antibody
- JV18 antibody
- JV18-1 antibody
- JV181 antibody
- MAD antibody
- MAD homolog 2 antibody
- MAD Related Protein 2 antibody
- Mad-related protein 2 antibody
- MADH2 antibody
- MADR2 antibody
- MGC22139 antibody
- MGC34440 antibody
- Mother against DPP homolog 2 antibody
- Mothers against decapentaplegic homolog 2 antibody
- Mothers against decapentaplegic, Drosophila, homolog of, 2 antibody
- Mothers against DPP homolog 2 antibody
- OTTHUMP00000163489 antibody
- Sma and Mad related protein 2 antibody
- Sma- and Mad-related protein 2 MAD antibody
- SMAD 2 antibody
- SMAD family member 2 antibody
- SMAD, mothers against DPP homolog 2 antibody
- SMAD2 antibody
- SMAD2_HUMAN antibody
see all

Western blot - Anti-Smad2 (phospho S250) antibody [EPR26263-58] (AB300079)

All lanes : Anti-Smad2 (phospho S250) antibody [EPR26263-58] (ab300079) at 1/1000 dilution

Lane 1 : Untreated parental HeLa (human cervix adenocarcinoma epithelial cell) whole cell lysate
Lane 2 : Parental HeLa treated with 0.2µM PMA for 0.5h whole cell lysate
Lane 3 : Untreated SAMD2 KO Hela whole cell lysate
Lane 4 : SAMD2 KO Hela treated with 0.2µM PMA for 0.5h whole cell lysate

Lysates/proteins at 30 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution

Predicted band size: 52 kDa
Observed band size: 62 kDa why is the actual band size different from the predicted?

Exposure time: 10 seconds

5% NFDM/TBST was used as blocking and diluting buffer.
Western blot - Anti-Smad2 (phospho S250) antibody [EPR26263-58] (AB300079)

All lanes : Anti-Smad2 (phospho S250) antibody [EPR26263-58] (ab300079) at 1/1000 dilution

Lane 1 : Untreated NIH/3T3 (Mouse embryonic fibroblast) whole cell lysate
Lane 2 : NIH/3T3 treated with 0.2µM PMA for 0.5h whole cell lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

Predicted band size: 52 kDa
Observed band size: 65 kDa why is the actual band size different from the predicted?

Exposure time: 180 seconds

5% NFDM/TBST was used as blocking and diluting buffer.
Western blot - Anti-Smad2 (phospho S250) antibody [EPR26263-58] (AB300079)

All lanes : Anti-Smad2 (phospho S250) antibody [EPR26263-58] (ab300079) at 1/1000 dilution

Lane 1 : Untreated C6 (rat glial tumor cell) whole cell lysate
Lane 2 : C6 treated with 0.2µM PMA for 0.5h whole cell lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

Predicted band size: 52 kDa
Observed band size: 65 kDa why is the actual band size different from the predicted?

Exposure time: 180 seconds

5% NFDM/TBST was used as blocking and diluting buffer.

Bands around and below the 50-kDa marker could be degradation fragments.
Western blot - Anti-Smad2 (phospho S250) antibody [EPR26263-58] (AB300079)

All lanes : Anti-Smad2 (phospho S250) antibody [EPR26263-58] (ab300079) at 1/1000 dilution

Lane 1 : HeLa (human cervix adenocarcinoma epithelial cell) treated with 0.2µM PMA for 0.5h (Untreated membrane) whole cell lysate
Lane 2 : HeLa treated with 0.2µM PMA for 0.5h (phosphatase treated membrane) whole cell lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

Predicted band size: 52 kDa
Observed band size: 65 kDa why is the actual band size different from the predicted?

Exposure time: 180 seconds

5% NFDM/TBST was used as blocking and diluting buffer.
Immunocytochemistry/ Immunofluorescence - Anti-Smad2 (phospho S250) antibody [EPR26263-58] (AB300079)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized HeLa (human cervix adenocarcinoma epithelial cell) cells lebeling Smad2 (phospho S250) with ab300079 at 1/50 (10.8 µg/ml) dilution, followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) preadsorbed antibody at 1/1000 (2 µg/mL) dilution (Green). Confocal image showing increased cytoplasm and nuclear staining in HeLa cells treated with PMA (200 nM ) for 30 min. ab195889 Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor^® 594) was used to counterstain tubulin at 1/200 (2.5 µg/ml) dilution (Red). The Nuclear counterstain was DAPI (Blue).

Secondary antibody only control: Secondary antibody is ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) preadsorbed at 1/1000 (2 µg/mL) dilution.
Immunoprecipitation - Anti-Smad2 (phospho S250) antibody [EPR26263-58] (AB300079)

Smad2 (phospho S250) was immunoprecipitated from 0.35 mg HeLa (human cervix adenocarcinoma epithelial cell) treated with 0.2µM PMA for 0.5h whole cell lysate with ab300079 at 1/30 dilution (2 µg in 0.35 mg lysates). Western blot was performed on the immunoprecipitate using ab300079 at 1/1000 dilution. VeriBlot for IP secondary antibody (HRP)(ab131366) was used at 1/5000 dilution.

Lane 1: HeLa (human cervix adenocarcinoma epithelial cell) treated with 0.2µM PMA for 0.5h whole cell lysate 10 µg (Inset)

Lane 2: ab300079 in HeLa treated with 0.2µM PMA for 0.5h whole cell lysate

Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab300079 in HeLa treated with 0.2µM PMA for 0.5h whole cell lysate

Blocking and dilution buffer and concentration: 5% NFDM/TBST.

Exposure time: 10 seconds
Immunoprecipitation - Anti-Smad2 (phospho S250) antibody [EPR26263-58] (AB300079)

Smad2 (phospho S250) was immunoprecipitated from 0.35 mg NIH/3T3 treated with 0.2µM PMA for 0.5h whole cell lysate with ab300079 at 1/30 dilution (2µg in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab300079 at 1/1000 dilution. VeriBlot for IP secondary antibody (HRP)(ab131366) was used at 1/5000 dilution.

Lane 1: NIH/3T3 treated with 0.2µM PMA for 0.5h whole cell lysate 20 µg (Inset)

Lane 2: ab300079 in NIH/3T3 treated with 0.2µM PMA for 0.5h whole cell lysate

Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab300079 in NIH/3T3 whole cell lysate

Blocking and dilution buffer and concentration: 5% NFDM/TBST.

Exposure time: 10 seconds
Dot Blot - Anti-Smad2 (phospho S250) antibody [EPR26263-58] (AB300079)

Dot blot analysis of Smad2 (phospho S250) using ab300079 at 1:1000 (0.54 µg/ml), followed by a Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (ab97051) at 1:100,000 dilution.

Lane 1: Smad2 (phospho S245+S250+S255) peptide a
Lane 2: Smad2 (phospho S245+S250+S255) peptide b
Lane 3: Smad2 (phospho S245+S250) peptide
Lane 4: Smad2 (phospho S245+S255) peptide
Lane 5: Smad2 (phospho S250+S255) peptide
Lane 6: Smad2 (phospho S245) peptide

Lane 7: Smad2 (phospho S250) peptide
Lane 8: Smad2 (phospho S255) peptide
Lane 9: Smad2 non-phospho peptide

Exposure time: 3 minutes

Blocking and diluting buffer and concentration: 5% NFDM/TBST
Anti-Smad2 (phospho S250) antibody [EPR26263-58] (AB300079)

To our knowledge, customised protocols are not required for this product. Please try the standard protocols listed below and let us know how you get on.

Click here to view the general protocols

SDS download

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Publishing research using ab300079? Please let us know so that we can cite the reference in this datasheet.

ab300079 has not yet been referenced specifically in any publications.

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Key features and details

Overview

Product name

Description

Host species

Tested applications

Species reactivity

Immunogen

Positive control

General notes

Properties

Form

Storage instructions

Storage buffer

Purity

Clonality

Clone number

Isotype

Research areas

Associated products

Alternative Versions

Related Products

Applications

The Abpromise guarantee

Target

Function

Tissue specificity

Sequence similarities

Post-translationalmodifications

Cellular localization

Database links

Alternative names

Images

Protocols

Datasheets and documents

SDS download

Datasheet download

Certificate of Compliance

References (0)

Customer reviews and Q&As

Post-translational
modifications