Anti-Smad4 antibody [EPR22589-112] (ab230815)
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
- Suitable for: IP, WB, ICC/IF, Flow Cyt
- Knockout validated
- Reacts with: Mouse, Rat, Human
Overview
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Product name
Anti-Smad4 antibody [EPR22589-112]
See all Smad4 primary antibodies -
Description
Rabbit monoclonal [EPR22589-112] to Smad4 -
Host species
Rabbit -
Tested Applications & Species
See all applications and species dataApplication Species Flow Cyt HumanICC/IF HumanIP MouseHumanWB MouseHuman -
Immunogen
Synthetic peptide. This information is proprietary to Abcam and/or its suppliers.
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Positive control
- WB: HAP1, HeLa, HCT116, HepG2, RAW264.7, HEK-293 and NIH/3T3 whole cell lysates; human fetal lung, mouse embryo, mouse lung and rat lung tissue lysates. Flow Cyt: WT HAP1 cells. ICC/IF: HeLa cell line treated with TGF-beta. IP: HeLa and NIH/3T3 whole cell lysates.
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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
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
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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: 7.2
Preservative: 0.01% Sodium azide
Constituents: PBS, 40% Glycerol (glycerin, glycerine), 0.05% BSA -
Concentration information loading...
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Purity
Protein A purified -
Clonality
Monoclonal -
Clone number
EPR22589-112 -
Isotype
IgG -
Research areas
Images
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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
Observed band size: 60 kDaBlocking/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.
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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.
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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.
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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.
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All lanes : Anti-Smad4 antibody [EPR22589-112] (ab230815) at 1/1000 dilution
Lane 1 : RAW264.7 (mouse Abelson murine leukemia virus-induced tumor macrophage) whole cell lysate
Lane 2 : NIH/3T3 (mouse embryonic fibroblast) 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: 60 kDa
Exposure time: 48 secondsBlocking/Dilution buffer: 5% NFDM/TBST.
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All lanes : Anti-Smad4 antibody [EPR22589-112] (ab230815) at 1/1000 dilution
Lane 1 : HEK-293 (human embryonic kidney epithelial cell) whole cell lysate
Lane 2 : HeLa (human cervix adenocarcinoma epithelial cell) 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: 60 kDa
Observed band size: 60 kDaBlocking/Dilution buffer: 5% NFDM/TBST.
Exposure time: Lane 1: 15 secs; Lane 2: 7.75 secs.
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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.
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