Astana Biomed Group, an authorized Abcam distributor in Central Asia

Anti-Smad4 antibody [EPR22589-112] - BSA and Azide free (ab254290)

Key features and details

  • Produced recombinantly (animal-free) for high batch-to-batch consistency and long term security of supply
  • Rabbit monoclonal [EPR22589-112] to Smad4 - BSA and Azide free
  • Suitable for: IP, Flow Cyt, ICC/IF, WB
  • Knockout validated
  • Reacts with: Mouse, Rat, Human

Overview

  • Product name

    Anti-Smad4 antibody [EPR22589-112] - BSA and Azide free
    See all Smad4 primary antibodies

  • Description

    Rabbit monoclonal [EPR22589-112] to Smad4 - BSA and Azide free

  • Host species

    Rabbit

  • Tested applications

    Suitable for: IP, Flow Cyt, ICC/IF, WBmore details
    Unsuitable for: ChIP or IHC-P

  • Species reactivity

    Reacts with: Mouse, Rat, Human

  • Immunogen

    Recombinant fragment. This information is proprietary to Abcam and/or its suppliers.

  • Positive control

    • WB: HAP1, HeLa, HCT116 and HepG2 whole cell lysates. Human fetal lung, mouse embryo, mouse lung and rat lung tissue lysate. Flow Cyt: WT HAP1 cells. ICC/IF: HeLa cell line treated with TGF-beta.IP: HeLa and NIH/3T3 whole cell lysates.

  • General notes

    Ab254290 is the carrier-free version of ab230815. 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

    ab254290 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.

    Reproducibility is key to advancing scientific discovery and accelerating scientists’ next breakthrough.

    Abcam is leading the way with our range of recombinant antibodies, knockout-validated antibodies and knockout cell lines, all of which support improved reproducibility.

    We are also planning to innovate the way in which we present recommended applications and species on our product datasheets, so that only applications & species that have been tested in our own labs, our suppliers or by selected trusted collaborators are covered by our Abpromise™ guarantee.

    In preparation for this, we have started to update the applications & species that this product is Abpromise guaranteed for.

    We are also updating the applications & species that this product has been “predicted to work with,” however this information is not covered by our Abpromise guarantee.

    Applications & species from publications and Abreviews that have not been tested in our own labs or in those of our suppliers are not covered by the Abpromise guarantee.

    Please check that this product meets your needs before purchasing. If you have any questions, special requirements or concerns, please send us an inquiry and/or contact our Support team ahead of purchase. Recommended alternatives for this product can be found below, as well as customer reviews and Q&As.

Images

  • Western blot - Anti-Smad4 antibody [EPR22589-112] - BSA and Azide free (ab254290)
    Western blot - Anti-Smad4 antibody [EPR22589-112] - BSA and Azide free (ab254290)
    All lanes : Anti-Smad4 antibody [EPR22589-112] (ab230815) at 1/1000 dilution

    Lane 1 : Smad4 knockout HAP1 whole cell lysate
    Lane 2 : Wild-type HAP1 whole cell lysate
    Lane 3 : HeLa (human cervix adenocarcinoma epithelial cell), whole cell lysate
    Lane 4 : HCT116 (human colorectal carcinoma epithelial cell) whole cell lysate
    Lane 5 : HepG2 (human hepatocellular carcinoma epithelial cell) whole cell lysate
    Lane 6 : Human fetal lung tissue lysate
    Lane 7 : Mouse embryo tissue lysate
    Lane 8 : Mouse lung tissue lysate
    Lane 9 : Rat lung tissue 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: 60 kDa



    Blocking/Dilution buffer: 5% NFDM/TBST.

    Exposure Time:  Lanes 1-6: 15 secs; Lanes 7-8: 114 secs; Lane 9: 3 mins.

    ab230815 was shown to specifically react with Smad4 in wild-type HAP1 cells as signal was lost in Smad4 knockout cells. Wild-type and Smad4 knockout samples were subjected to SDS-PAGE. ab230815 and ab181602 (Rabbit anti-GAPDH loading control) were incubated 1 hour at room temperature at 1/1000 dilution and 1/200,000 dilution respectively. Blots were developed with Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (ab97051) secondary antibody at 1/100,000 dilution for 1 hour at room temperature before imaging.

     This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab230815).

  • Immunocytochemistry/ Immunofluorescence - Anti-Smad4 antibody [EPR22589-112] - BSA and Azide free (ab254290)
    Immunocytochemistry/ Immunofluorescence - Anti-Smad4 antibody [EPR22589-112] - BSA and Azide free (ab254290)

    Immunofluorescent analysis of HeLa (human cervix adenocarcinoma epithelial cell) cells labeling Smad4 with ab230815 at a 1/100 dilution (green). Cells were fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100. Confocal image showing mainly nuclear staining in HeLa cell line treated with TGF-beta (10ng/ml) for 1 h. AlexaFluor®488 Goat anti-Rabbit (ab150077) was used secondary antibody at 1/1000 dilution, this was also used on its own as a control. DAPI was used as a nuclear counterstain (blue). Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor® 594, ab195889) was used as a counterstain at 1/200 dilution.

    This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab230815).

  • Flow Cytometry - Anti-Smad4 antibody [EPR22589-112] - BSA and Azide free (ab254290)
    Flow Cytometry - Anti-Smad4 antibody [EPR22589-112] - BSA and Azide free (ab254290)

    Flow cytometric analysis of 4% paraformaldehyde-fixed Smad4 KO HAP1 (Smad4 knockout human chronic myelogenous leukemia near-haploid cell line, Left) / WT HAP1 (human chronic myelogenous leukemia near-haploid cell line, Right) cell line labeling Smad4 with ab230815 at 1/500 (Red) compared with a Rabbit monoclonal IgG (ab172730) (Black) and an unlabeled control (cells without incubation with primary antibody and secondary antibody) (Blue). Goat anti rabbit IgG (Alexa Fluor® 488, ab150077) was used as secondary antibody at 1/2000 dilution.

    This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab230815).

  • Immunoprecipitation - Anti-Smad4 antibody [EPR22589-112] - BSA and Azide free (ab254290)
    Immunoprecipitation - Anti-Smad4 antibody [EPR22589-112] - BSA and Azide free (ab254290)

    Smad4 was immunoprecipitated from 0.35mg of HeLa (human cervix adenocarcinoma epithelial cell) whole cell lysate with ab230815 at 1/30 dilution. Western Blot was performed from the immunoprecipitate using ab230815 at 1/1000 dilution (0.5 μg/ml).

    VeriBlot for IP Detection Reagent (HRP) (ab131366), was used as secondary antibody at 1/5000 dilution.

    Lane 1: HeLa whole cell lysate 10μg (Input).

    Lane 2: ab230815 IP in HeLa whole cell lysate

    Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab230815 in HeLa whole cell lysate

    Blocking/Dilution buffer: 5% NFDM/TBST.

    Exposure time: 30 seconds

    Lysate was freshly prepared and IP test was done immediately to avoid protein degradation. Lysate incubation time was shortened from overnight to 2h.

    This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab230815).

  • Immunoprecipitation - Anti-Smad4 antibody [EPR22589-112] - BSA and Azide free (ab254290)
    Immunoprecipitation - Anti-Smad4 antibody [EPR22589-112] - BSA and Azide free (ab254290)

    Smad4 was immunoprecipitated from 0.35mg of NIH/3T3 (mouse embryonic fibroblast) whole cell lysate with ab230815 at 1/30 dilution. Western Blot was performed from the immunoprecipitate using ab230815 at 1/1000 dilution (0.5 μg/ml).

    VeriBlot for IP Detection Reagent (HRP) (ab131366), was used as secondary antibody at 1/5000 dilution.

    Lane 1: NIH/3T3 whole cell lysate 10μg (Input).

    Lane 2: ab230815 IP in NIH/3T3 whole cell lysate

    Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab230815 in NIH/3T3 whole cell lysate

    Blocking/Dilution buffer: 5% NFDM/TBST.

    Exposure time: 10 seconds

    Lysate was freshly prepared and IP test was done immediately to avoid protein degradation. Lysate incubation time was shortened from overnight to 2h.

    This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab230815).

  • Anti-Smad4 antibody [EPR22589-112] - BSA and Azide free (ab254290)
    Anti-Smad4 antibody [EPR22589-112] - BSA and Azide free (ab254290)

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