Overview

  • Product name
    Anti-Endothelin 1 antibody [TR.ET.48.5]
    See all Endothelin 1 primary antibodies
  • Description
    Mouse monoclonal [TR.ET.48.5] to Endothelin 1
  • Host species
    Mouse
  • Specificity
    Immunohistochemical staining of ET-1 in human corpus cavernosum tissue with this antibody results in staining of endothelial cells. Radioimmune assays can be used to concentrate ET-1 in solution (e.g. serum/plasma, milk, urine).
  • Tested applications
    Suitable for: RIA, IP, Inhibition Assay, ELISA, Flow Cyt, ICC/IF, IHC-Fr, IHC-P, WBmore details
  • Species reactivity
    Reacts with: Mouse, Rat, Sheep, Dog, Human
  • Immunogen

    Full length protein corresponding to Human Endothelin 1 conjugated to Keyhole Limpet Haemocyanin (KLH).

  • Epitope
    Studies suggest that this antibody binds to an epitope in the region of ET-1 represented by amino acids 8-16.
  • Positive control
    • human bowel

Properties

Applications

Our Abpromise guarantee covers the use of ab2786 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
RIA 1/25000.
IP Use at an assay dependent concentration.
Inhibition Assay Use at an assay dependent concentration.
ELISA Use at an assay dependent concentration.
Flow Cyt Use at an assay dependent concentration.

ab170190 - Mouse monoclonal IgG1, is suitable for use as an isotype control with this antibody.

ICC/IF 1/200 - 1/1000.
IHC-Fr 1/250.
IHC-P 1/250.
WB Use at an assay dependent concentration. Predicted molecular weight: 24 kDa.

Target

Images

  • ab2786 labelling Endothelin 1 (green) in the secretion of HeLa cells (right) compared with a negative control (left) by Immunocytochemistry/Immunofluorescence. Formalin-fixed cells were permeabilized with 0.1% Triton X-100 in TBS for 5-10 minutes and blocked with 3% BSA-PBS for 30 minutes at room temperature. Cells were incubated with the primary antibody (1:200 in 3% BSA-PBS) overnight at 4 ºC. A DyLight 488-conjugated Goat anti-mouse IgG (H+L) was used as the secondary antibody. Red (phalloidin) - F-actin, Blue (DAPI) - nuclei. Images were taken at a magnification of 60x.

  • Anti-Endothelin 1 antibody [TR.ET.48.5] (ab2786) at 1/500 dilution + PC12 cell lysate at 25 µg

    Predicted band size: 24 kDa
    Observed band size: 30 kDa
    why is the actual band size different from the predicted?

  • Flow cytometry analysis of Endothelin 1 showing positive staining in the cytoplasm of 293T cells compared to an isotype control (blue). Cells were harvested, adjusted to a concentration of 1-5x10^6 cells/ml, fixed with 2% paraformaldehyde, washed with PBS, and incubated with ab2786 (0.5 ug/test) for 60 min at room temperature. Cells were then blocked in a solution of 2% BSA-PBS for 30 min at room temperature, incubated for 40 min at room temperature in the dark using a Dylight 488-conjugated goat anti-mouse IgG (H+L) secondary antibody, and re-suspended in PBS for FACS analysis.

  • ab2786 labelling Endothelin 1 (green) in the secretion of PC12 cells (right) compared with a negative control (left) by Immunocytochemistry/Immunofluorescence. Formalin-fixed cells were permeabilized with 0.1% Triton X-100 in TBS for 5-10 minutes and blocked with 3% BSA-PBS for 30 minutes at room temperature. Cells were incubated with the primary antibody (1:200 in 3% BSA-PBS) overnight at 4 ºC. A DyLight 488-conjugated Goat anti-mouse IgG (H+L) was used as the secondary antibody. Red (phalloidin) - F-actin, Blue (DAPI) - nuclei. Images were taken at a magnification of 60x.

  • Flow cytometry analysis of Endothelin 1 showing positive staining in the cytoplasm of HepG2 cells compared to an isotype control (blue). Cells were harvested, adjusted to a concentration of 1-5x10^6 cells/ml, fixed with 2% paraformaldehyde, washed with PBS, and incubated with ab2786 (0.5 ug/test) for 60 min at room temperature. Cells were then blocked in a solution of 2% BSA-PBS for 30 min at room temperature, incubated for 40 min at room temperature in the dark using a Dylight 488-conjugated goat anti-mouse IgG (H+L) secondary antibody, and re-suspended in PBS for FACS analysis.

  • ab2786 labelling Endothelin 1 (green) in the secretion of HUVEC cells (right) compared with a negative control (left) by Immunocytochemistry/Immunofluorescence. Formalin-fixed cells were permeabilized with 0.1% Triton X-100 in TBS for 5-10 minutes and blocked with 3% BSA-PBS for 30 minutes at room temperature. Cells were incubated with the primary antibody (1:200 in 3% BSA-PBS) overnight at 4 ºC. A DyLight 488-conjugated Goat anti-mouse IgG (H+L)  was used as the secondary antibody. Red (phalloidin) - F-actin, Blue (DAPI) - nuclei. Images were taken at a magnification of 60x.

  • Flow cytometry analysis of Endothelin 1 showing positive staining in the cytoplasm of 3T3 cells compared to an isotype control (blue). Cells were harvested, adjusted to a concentration of 1-5x10^6 cells/ml, fixed with 2% paraformaldehyde, washed with PBS, and incubated with ab2786 (0.5 ug/test) for 60 min at room temperature. Cells were then blocked in a solution of 2% BSA-PBS for 30 min at room temperature, incubated for 40 min at room temperature in the dark using a Dylight 488-conjugated goat anti-mouse IgG (H+L) secondary antibody, and re-suspended in PBS for FACS analysis.

  • Figure 1 and Figure 2 show immunolocalization of ET-1 in human bowel using ab2786.

References

This product has been referenced in:
  • Li JR  et al. Fasudil improves endothelial dysfunction in rats exposed to chronic intermittent hypoxia through RhoA/ROCK/NFATc3 pathway. PLoS One 13:e0195604 (2018). Read more (PubMed: 29641598) »
  • Hsu PL  et al. Ganoderma Triterpenoids Exert Antiatherogenic Effects in Mice by Alleviating Disturbed Flow-Induced Oxidative Stress and Inflammation. Oxid Med Cell Longev 2018:3491703 (2018). Read more (PubMed: 29849882) »
See all 26 Publications for this product

Customer reviews and Q&As

1-8 of 8 Abreviews or Q&A

Answer

Thank you for your reply and further enqjuiry. I am sorry to confirm that the IHC image provided on the datasheet is the only one available at present. There are also 6 publications cited on the datasheet which have used this antibody. Ican suggest to review the data within these for further reassurance: Mayyas F et al. Dietary 3 fatty acids modulate the substrate for post-operative atrial fibrillation in a canine cardiac surgery model. Cardiovasc Res 89:852-61 (2011). Dog. PubMed: 21123218 Chen CL et al. Increased endothelin 1 expression in adult-onset minimal change nephropathy with acute renal failure. Am J Kidney Dis 45:818-25 (2005). PubMed: 15861346 Nelson JB et al. Endothelin-1 production and decreased endothelin B receptor expression in advanced prostate cancer. Cancer Res 56:663-8 (1996). PubMed: 8630991 Traish AM et al. Monoclonal antibodies to human endothelin-1: characterization and utilization in radioimmunoassay and immunocytochemistry. Hybridoma 11:147-63 (1992). PubMed: 1607212 Rubanyi GM & Botelho LH Endothelins. FASEB J 5:2713-20 (1991). PubMed: 1916094 Saenz de Tejada I et al. Endothelin: localization, synthesis, activity, and receptor types in human penile corpus cavernosum. Am J Physiol 261:H1078-85 (1991). PubMed: 1656784 The cellular localisation of Endothelin 1 is 'secreted. Therefore, in tissue, it is likely to be localised between the cells. There may be some cytoplasmic staining of Endothelin that has not yet been secreted from the cells. I hope this will be helpful to you. If you have any further questions, please do not hesitate to contact us.

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Answer

Thank you for your message and for kindly providing the requested details. I am pleased to provide the following information: Endothelin -1 antibody ab2786 According to the image on the datasheet, this antibody has been successfully used in  human bowel samples, and I can suggest this would be a suitable positive control. According to the SwissProt/Uniprot protein database site, it is also expressed in the lung and  placental stem villi vessels and these would also be suitable positive controls.  As endothelin 1 is found in the smooth muscle tissue of blood vessels, it will be found in most tissue types. therefore, it would be difficult to suggest a negative control. I can recommend a literature search to obtain further information. Endothelin A Receptor antibody ab76259 According to the image on the datasheet, this antibody has been successfully used in  human cerebellum samples, and I can suggest this would be a suitable positive control. Its tissue specificity, from the SwissProt/Uniprot protein database link is as follows: Isoform 1, isoform 3 and isoform 4 are expressed in a variety of tissues, with highest levels in the aorta and cerebellum, followed by lung, atrium and cerebral cortex, lower levels in the placenta, kidney, adrenal gland, duodenum, colon, ventricle and liver but no expression in umbilical vein endothelial cells. Within the placenta, isoform 1, isoform 2, isoform 3 and isoform 4 are expressed in the villi and stem villi vessels.  Therefore, these tissues would also be suitable positive controls. Again, Endothelin A receptor it is quite widely expressed, and so suggesting a suitable negative control is difficult.   I hope this information is helpful to you. Should you have any further questions, please do not hesitate to contact us.

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Answer

Thank you for your enquiry. With necrotic tissue, it is very likely that the proteins are already being broken down and degraded by the enzymes that are already present. I am sorry it would be very difficult to say and guarantee how much Endothelin 1 or PDE5A would still be in the sample for detection by the antibody. If the proteins are released from the cell during necrosis, the localization of the protein may also be affected.  If the tissue is already necrotic before fixation, then there are no fixation methods that would specifically help in this case. I can suggest to try the usual fixation methods. I can suggest it would be important to consider including positive control tissue. For example: PDE5A antibody (ab14672) has been used successfully in rat human heart tissue. We do have the following slides available: ab4332  Heart (human): aortic valve normal tissue slides Applications: IHC-P, ISH   Endothelin 1 antibody (ab2786) has been used successfully in human bowel. We do have the following slides available: ab4327 Colon (human) normal tissue slides Applications: IHC-P, ISH    I hope this information will be helpful to you.  If you have any further questions, please do not hesitate to contact me.    

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Answer

Thank you for your enquiry. I'm afraid we have no data regarding the detection of PreproET or Big ET using this antibody at this time. Both ET-1 and Big ET are contained within PreproET. Therefore I would expect that this antibody would detect PreproET as well, however I have no data to support this hypothesis and therefore I cannot guarantee it will detect prepro and big ET. The antibody has been used in immunohistochemical staining of ET-1 in human corpus cavernosum tissue with this antibody resulting in staining of endothelial cells and it also has successfully been used in radioimmune assays to concentrate ET-1 in solution (e.g. serum/plasma, milk, urine). Please do not hesitate to contact me if I can be of further assistance,

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Question
Answer

Thank you for your enquiry. It is not possible to determine the antibody concentration for this product, since it is ascites, and ascites contains proteins besides the antibody of interest. Ascites fluid is clarified by centrifugation to remove the lipid layer and the cell pellet. It contains specific antibody as well as other host serum proteins including immunoglobulins. While the total protein concentration is ~20 mg/ml, the specific antibody concentration range is 1-10 mg/ml. An exact antibody concentration is only relevant for purified antibodies. I hope this information helps, please do not hesitate to contact us if you need any more advice or information.

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Answer

I am very sorry. I misunderstood your question. Yes, using mouse IgG1 will allow you to determine what antibodies found in mouse IgG1 will bind to your slides. If you see some binding then this is background that should not be confused with your ET-1 positive staining. Another control would be to omit the primary antibody so that you can see what non-specific staining is present from the secondary antibody. Finally, since studies suggest that this antibody binds to an epitope in the region of ET-1 represented by amino acids 8-16, a final control would be using the peptide of these amino acids in a blocking experiment. We do not have this peptide available, but it may be commercially available or could be synthesized. I hope this information helps, please do not hesitate to contact us if you need any more advice or information,

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Answer

Thank you for your enquiry. Ab2786 detects ET-1 in human tissues. This antibody shows little cross-reactivity to related proteins. The antigen is purified human endothelin-1. Studies suggest that this antibody binds to an epitope in the region of ET-1 represented by amino acids 8-16. We hope this will help,

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Answer

Thank you for your email. Since ab2786 has not yet been tested in Western blotting (to our knowledge), it's tricky to say what could be going on. At this point I would suggest varying the concentrations of both your primary and secondary antibodies, make sure to run a secondary antibody control (no primary), and try running different sample types. Also, make sure to run a positive control. Please let us know how you get on and if you need further information, please contact us again and we will gladly assist you.

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