Astana Biomed Group, an authorized Abcam distributor in Central Asia

Anti-Smad2 (phospho T8) + Smad3 (phospho T8) antibody [EPR23682-64] - BSA and Azide free (ab276140)

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) - BSA and Azide free
  • Suitable for: WB, ICC, Dot blot, IP
  • Reacts with: Mouse, Rat, Human

Overview

  • Product name

    Anti-Smad2 (phospho T8) + Smad3 (phospho T8) antibody [EPR23682-64] - BSA and Azide free

  • Description

    Rabbit monoclonal [EPR23682-64] to Smad2 (phospho T8) + Smad3 (phospho T8) - BSA and Azide free

  • Host species

    Rabbit

  • Tested applications

    Suitable for: WB, ICC, Dot blot, IPmore 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.

    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: HaCaT and RAW264.7 cells. IP: HaCaT whole cell lysate.

  • General notes

    ab276140 is the carrier-free version of ab254407. This format is designed for use in antibody labeling, including fluorochromes, metal isotopes, oligonucleotides, enzymes.

    Our carrier-free formats are supplied in a buffer free of BSA, sodium azide and glycerol for higher conjugation efficiency.

    Use our conjugation kits for antibody conjugates that are ready-to-use in as little as 20 minutes with

    ab276140 is compatible with the Maxpar® Antibody Labeling Kit from Fluidigm.

    Maxpar® is a trademark of Fluidigm Canada Inc.

    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.

Properties

Images

  • Immunocytochemistry - Anti-Smad2 (phospho T8) + Smad3 (phospho T8) antibody [EPR23682-64] - BSA and Azide free (ab276140)
    Immunocytochemistry - Anti-Smad2 (phospho T8) + Smad3 (phospho T8) antibody [EPR23682-64] - BSA and Azide free (ab276140)

    This data was developed using ab254407, the same antibody clone in a different buffer formulation.

    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] - BSA and Azide free (ab276140)
    Western blot - Anti-Smad2 (phospho T8) + Smad3 (phospho T8) antibody [EPR23682-64] - BSA and Azide free (ab276140)
    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), Peroxidase conjugated (ab97051) at 1/100000 dilution

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



    This data was developed using ab254407, the same antibody clone in a different buffer formulation.

    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] - BSA and Azide free (ab276140)
    Immunoprecipitation - Anti-Smad2 (phospho T8) + Smad3 (phospho T8) antibody [EPR23682-64] - BSA and Azide free (ab276140)

    This data was developed using ab254407, the same antibody clone in a different buffer formulation.

    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] - BSA and Azide free (ab276140)
    Dot Blot - Anti-Smad2 (phospho T8) + Smad3 (phospho T8) antibody [EPR23682-64] - BSA and Azide free (ab276140)

    This data was developed using ab254407, the same antibody clone in a different buffer formulation.

    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 - Anti-Smad2 (phospho T8) + Smad3 (phospho T8) antibody [EPR23682-64] - BSA and Azide free (ab276140)
    Immunocytochemistry - Anti-Smad2 (phospho T8) + Smad3 (phospho T8) antibody [EPR23682-64] - BSA and Azide free (ab276140)

    This data was developed using ab254407, the same antibody clone in a different buffer formulation.

    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] - BSA and Azide free (ab276140)
    Western blot - Anti-Smad2 (phospho T8) + Smad3 (phospho T8) antibody [EPR23682-64] - BSA and Azide free (ab276140)
    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?



    This data was developed using ab254407, the same antibody clone in a different buffer formulation.

    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] - BSA and Azide free (ab276140)
    Western blot - Anti-Smad2 (phospho T8) + Smad3 (phospho T8) antibody [EPR23682-64] - BSA and Azide free (ab276140)
    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?



    This data was developed using ab254407, the same antibody clone in a different buffer formulation.

    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] - BSA and Azide free (ab276140)
    Western blot - Anti-Smad2 (phospho T8) + Smad3 (phospho T8) antibody [EPR23682-64] - BSA and Azide free (ab276140)
    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?



    This data was developed using ab254407, the same antibody clone in a different buffer formulation.

    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] - BSA and Azide free (ab276140)
    Anti-Smad2 (phospho T8) + Smad3 (phospho T8) antibody [EPR23682-64] - BSA and Azide free (ab276140)

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