Recombinant Anti-Smad2 (phospho T8) + Smad3 (phospho T8) antibody [EPR23682-64] (ab254407)

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Immunocytochemistry/ Immunofluorescence - Anti-Smad2 (phospho T8) + Smad3 (phospho T8) antibody [EPR23682-64] (ab254407)

Key features and details

Produced recombinantly (animal-free) for high batch-to-batch consistency and long term security of supply
Rabbit monoclonal [EPR23682-64] to Smad2 (phospho T8) + Smad3 (phospho T8)
Suitable for: ICC/IF, Dot blot, IP, WB
Reacts with: Mouse, Rat, Human

Carrier Free

Product name

Anti-Smad2 (phospho T8) + Smad3 (phospho T8) antibody [EPR23682-64]
Description

Rabbit monoclonal [EPR23682-64] to Smad2 (phospho T8) + Smad3 (phospho T8)
Host species

Rabbit
Tested applications

Suitable for: ICC/IF, Dot blot, IP, WBmore details
Unsuitable for: Flow Cyt 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: RAW264.7, PC-12, HaCaT, and HaCaT ( treated with 100nM calyculin A for 30 min) whole cell lysates; Human liver cancer tissue lysate; Mouse lung and liver tissue lysates. ICC/IF: HaCaT and RAW264.7 cells. IP: HaCaT whole cell lysate.
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

Preservative: 0.01% Sodium azide
Constituents: 59.94% PBS, 40% Glycerol (glycerin, glycerine), 0.05% BSA
Concentration information loading...
Purity

Protein A purified
Clonality

Monoclonal
Clone number

EPR23682-64
Isotype

IgG
Research areas

Alternative Versions
- Anti-Smad2 (phospho T8) + Smad3 (phospho T8) antibody [EPR23682-64] - BSA and Azide free (ab276140)
Compatible Secondaries
Isotype control
- Rabbit IgG, monoclonal [EPR25A] - Isotype Control (ab172730)
Related Products
- Anti-Smad2 + Smad3 antibody [EPR19557-4] - ChIP Grade (ab202445)

The Abpromise guarantee

Our Abpromise guarantee covers the use of ab254407 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
ICC/IF		1/50.
Dot blot		1/1000.
IP		1/30.
WB		1/1000. Detects a band of approximately 55, 60 kDa.

Notes
ICC/IF 1/50.
Dot blot 1/1000.
IP 1/30.
WB 1/1000. Detects a band of approximately 55, 60 kDa.

Application notes

Is unsuitable for Flow Cyt or IHC-P.

Cellular localization

Smad2: 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. Smad3: Cytoplasm. Nucleus. Cytoplasmic and nuclear in the absence of TGF-beta. On TGF-beta stimulation, migrates to the nucleus when complexed with SMAD4 (PubMed:15799969). Through the action of the phosphatase PPM1A, released from the SMAD2/SMAD4 complex, and exported out of the nucleus by interaction with RANBP1 (PubMed:16751101, PubMed:19289081). Co-localizes with LEMD3 at the nucleus inner membrane (PubMed:15601644). MAPK-mediated phosphorylation appears to have no effect on nuclear import (PubMed:19218245). PDPK1 prevents its nuclear translocation in response to TGF-beta (PubMed:17327236).
Database links
- Entrez Gene: 4087 Human
- Entrez Gene: 4088 Human
- Entrez Gene: 17126 Mouse
- Entrez Gene: 17127 Mouse
- Entrez Gene: 25631 Rat
- Entrez Gene: 29357 Rat
- Omim: 601366 Human
- Omim: 603109 Human
- SwissProt: P84022 Human
- SwissProt: Q15796 Human
- SwissProt: Q62432 Mouse
- SwissProt: Q8BUN5 Mouse
- SwissProt: O70436 Rat
- SwissProt: P84025 Rat
- Unigene: 12253 Human
- Unigene: 705764 Human
- Unigene: 727986 Human
- Unigene: 742270 Human
- Unigene: 391091 Mouse
- Unigene: 7320 Mouse
- Unigene: 10636 Rat
- Unigene: 2755 Rat
see all

Immunocytochemistry/ Immunofluorescence - Anti-Smad2 (phospho T8) + Smad3 (phospho T8) antibody [EPR23682-64] (ab254407)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized RAW 264.7 cells labelling Smad2 (phospho T8) + Smad3 (phospho T8) with ab254407 at 1/50 (9.34 ug/ml) dilution, followed by ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) antibody at 1/1000 dilution (Green). Confocal image showing weak cytoplasmic and nuclear staining in RAW 264.7 cells, while strong cytoplasmic and weak nuclear staining in RAW 264.7 cells treated with calyculin A (100 nM) for 30 mins is observed. ab195889 Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor^® 594) was used to counterstain tubulin at 1/200 dilution (Red). The Nuclear counterstain was DAPI (Blue).

Secondary antibody only control: Secondary antibody is ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) at 1/1000 dilution.
Western blot - Anti-Smad2 (phospho T8) + Smad3 (phospho T8) antibody [EPR23682-64] (ab254407)

All lanes : Anti-Smad2 (phospho T8) + Smad3 (phospho T8) antibody [EPR23682-64] (ab254407) at 1/1000 dilution

Lane 1 : HaCaT (human skin keratinocyte), whole cell lysate at 10 µg
Lane 2 : HaCaT - phosphatase treated membrane, whole cell lysate at 10 µg
Lanes 3 & 5 : HaCaT whole cell lysate at 20 µg
Lane 4 : HaCaT treated with 100nM calycin A for 30 min, whole cell lysate at 20 µg
Lane 6 : HaCaT treated with /ml TGF beta1 for 24h, whole cell lysate at 20 µg

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

Observed band size: 55,60 kDa why is the actual band size different from the predicted?

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

The molecular weight observed is consistent with what has been described in the literature. (PMID: 25998442, 30666129).

Calyculin A is a known phosphatase inhibitor, which increased the level of pSmad2/3 (T8). The shifted band after treated with Calyculin A might be due to multiple phosphorylation events.

The down-regulation of pSmad2 (T8) is induced by TGF-beta1 treatment in HaCaT (PMID: 19201832).

Bands between 15-25kDa may be caused by degradation.

Exposure time: Lane 1, 2: 26 secondsLane 3, 4: 59 secondsLane 5, 6: 3 minutes
Immunoprecipitation - Anti-Smad2 (phospho T8) + Smad3 (phospho T8) antibody [EPR23682-64] (ab254407)

Smad2 (phospho T8) + Smad3 (phospho T8) was immunoprecipitated from 0.35 mg HaCaT (human skin keratinocyte), whole cell lysate with ab254407 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab254407 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP)(ab131366) was used at 1/5000 dilution.

Lane 1: HaCaT (human skin keratinocyte), whole cell lysate 10 ug

Lane 2: ab254407 IP in HaCaT whole cell lysate

Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab254407 in HaCaT whole cell lysate

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

Exposure time: 110 seconds

The molecular weight observed is consistent with what has been described in the literature. (PMID: 25998442, 30666129).
Dot Blot - Anti-Smad2 (phospho T8) + Smad3 (phospho T8) antibody [EPR23682-64] (ab254407)

Dot blot analysis of Smad2 (phospho T8) + Smad3 (phospho T8) using ab254407 at 1/1000 (0.467 ug/ml) dilution followed by a Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (ab97051) at 1/100,000 dilution.

Exposure time: 3 minutes.

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

Lane 1: Smad2/3 peptide (aa 6-14)

Lane 2: Smad2/3 (phospho T8) peptide (aa 2-10 )

Lane 3: Smad2/3 peptide (aa 2-14)
Immunocytochemistry/ Immunofluorescence - Anti-Smad2 (phospho T8) + Smad3 (phospho T8) antibody [EPR23682-64] (ab254407)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized HaCaT cells labelling Smad2 (phospho T8) + Smad3 (phospho T8) with ab254407 at 1/50 (9.34 ug/ml) dilution, followed by ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) antibody at 1/1000 dilution (Green). Confocal image showing weak cytoplasmic and nuclear staining in HaCaT cells, while strong cytoplasmic and weak nuclear staining in HaCaT cells treated with calyculin A (100 nM) for 30 mins is observed. ab195889 Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor^® 594) was used to counterstain tubulin at 1/200 dilution (Red). The Nuclear counterstain was DAPI (Blue).

Secondary antibody only control: Secondary antibody is ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) at 1/1000 dilution.
Western blot - Anti-Smad2 (phospho T8) + Smad3 (phospho T8) antibody [EPR23682-64] (ab254407)

All lanes : Anti-Smad2 (phospho T8) + Smad3 (phospho T8) antibody [EPR23682-64] (ab254407) at 1/1000 dilution

Lane 1 : RAW264.7 (mouse Abelson murine leukemia virus-induced tumor macrophage), whole cell lysate
Lane 2 : RAW264.7 - phosphatase treated membrane, whole cell lysate

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (ab97051) at 1/100000 dilution

Observed band size: 60 kDa why is the actual band size different from the predicted?

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

Exposure time: 26 seconds.
Western blot - Anti-Smad2 (phospho T8) + Smad3 (phospho T8) antibody [EPR23682-64] (ab254407)

All lanes : Anti-Smad2 (phospho T8) + Smad3 (phospho T8) antibody [EPR23682-64] (ab254407) at 1/1000 dilution

Lane 1 : PC-12 (rat adrenal gland pheochromocytoma), whole cell lysate
Lane 2 : PC-12 - phosphatase treated membrane, whole cell lysate

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (ab97051) at 1/100000 dilution

Observed band size: 60 kDa why is the actual band size different from the predicted?

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

The molecular weight observed is consistent with what has been described in the literature. (PMID: 25998442, 30666129)

Exposure time: 59 seconds.
Western blot - Anti-Smad2 (phospho T8) + Smad3 (phospho T8) antibody [EPR23682-64] (ab254407)

All lanes : Anti-Smad2 (phospho T8) + Smad3 (phospho T8) antibody [EPR23682-64] (ab254407) at 1/1000 dilution

Lane 1 : Human liver cancer tissue lysate
Lane 2 : Mouse lung tissue lysate
Lane 3 : Mouse liver tissue lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (ab97051) at 1/100000 dilution

Observed band size: 55,60 kDa why is the actual band size different from the predicted?

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

The molecular weight observed is consistent with what has been described in the literature. (PMID: 25998442, 30666129).

Bands between 15-25kDa may be caused by degradation.

Exposure time: 3 minutes.
Anti-Smad2 (phospho T8) + Smad3 (phospho T8) antibody [EPR23682-64] (ab254407)

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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Datasheet download

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Publishing research using ab254407? Please let us know so that we can cite the reference in this datasheet.

ab254407 has not yet been referenced specifically in any publications.

Customer reviews and Q&As

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Key features and details

Related conjugates and formulations

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

Compatible Secondaries

Isotype control

Related Products

Applications

The Abpromise guarantee

Target

Cellular localization

Database links

Images

Protocols

Datasheets and documents

SDS download

Datasheet download

Certificate of Compliance

References (0)

Customer reviews and Q&As