Anti-Angiopoietin 1 antibody (ab95230)

Rabbit polyclonal Angiopoietin 1 antibody. Validated in WB, ICC/IF and tested in Mouse, Sheep. Cited in 6 publication(s). Independently reviewed in 1 review(s).

Overview

  • Product name

    Anti-Angiopoietin 1 antibody
    See all Angiopoietin 1 primary antibodies
  • Description

    Rabbit polyclonal to Angiopoietin 1
  • Host species

    Rabbit
  • Tested applications

    Suitable for: ICC/IF, WBmore details
  • Species reactivity

    Reacts with: Mouse, Sheep
    Predicted to work with: Rat, Cow, Dog, Human, Pig
  • Immunogen

    Synthetic peptide corresponding to Mouse Angiopoietin 1 aa 1-100 conjugated to keyhole limpet haemocyanin.
    (Peptide available as ab109637, ab134973, ab9076)

  • Positive control

    • This antibody gave a positive signal in both E10 and E11 Mouse embryonic brain tissue lysates and MEF1 cell line.

Properties

Applications

Our Abpromise guarantee covers the use of ab95230 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 Use a concentration of 5 µg/ml.
WB Use a concentration of 1 µg/ml. Detects a band of approximately 53 kDa (predicted molecular weight: 57 kDa).

Target

Images

  • Anti-Angiopoietin 1 antibody (ab95230) at 1 µg/ml + E10 Mouse Embryo Brain Tissue Lysate at 10 µg

    Secondary
    Goat Anti-Rabbit IgG H&L (HRP) preadsorbed (ab97080) at 1/5000 dilution

    Developed using the ECL technique.

    Performed under reducing conditions.

    Predicted band size: 57 kDa
    Observed band size: 53 kDa
    why is the actual band size different from the predicted?
    Additional bands at: 95 kDa. We are unsure as to the identity of these extra bands.


    Exposure time: 20 minutes


    The band observed at 53 kDa could potentially be a cleaved form of Angiopoietin 1 due to the presence of a 19 amino acid signal peptide. Abcam recommends using milk as the blocking agent. Abcam welcomes customer feedback and would appreciate any comments regarding this product and the data presented above.
  • ICC/IF image of ab95230 stained MEF1 cells. The cells were 4% formaldehyde fixed (10 min) and then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1h to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab95230, 5µg/ml) overnight at +4°C. The secondary antibody (green) was ab96899, DyLight® 488 goat anti-rabbit IgG (H+L) used at a 1/250 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (red) at a 1/200 dilution for 1h. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43µM.
  • All lanes : Anti-Angiopoietin 1 antibody (ab95230) at 1/500 dilution

    Lane 1 : Sheep fetal extra-embryonic membrane purified protein at 50 µg
    Lane 2 : Sheep fetal extra-embryonic membrane purified protein at 30 µg
    Lane 3 : Sheep fetal extra-embryonic membrane purified protein at 20 µg
    Lane 4 : Sheep fetal extra-embryonic membrane purified protein at 10 µg

    Secondary
    All lanes : HRP-conjugated goat anti-rabbit IgG monoclonal at 1/5000 dilution

    Developed using the ECL technique.

    Performed under reducing conditions.

    Predicted band size: 57 kDa
    Observed band size: 57 kDa


    Exposure time: 90 seconds

    See Abreview

References

This product has been referenced in:

  • Lu Y  et al. Transcriptome Profiling of Neovascularized Corneas Reveals miR-204 as a Multi-target Biotherapy Deliverable by rAAVs. Mol Ther Nucleic Acids 10:349-360 (2018). WB . Read more (PubMed: 29499946) »
  • Gram A  et al. Angiopoietin expression in ovine corpora lutea during the luteal phase: Effects of nutrition, arginine and follicle stimulating hormone. Gen Comp Endocrinol 269:131-140 (2018). Read more (PubMed: 30195024) »
See all 8 Publications for this product

Customer reviews and Q&As

1-5 of 5 Abreviews or Q&A

Application
Western blot
Loading amount
50 µg
Gel Running Conditions
Reduced Denaturing (10 %)
Sample
Sheep Purified protein (fetal extra-embryonic membrane)
Specification
fetal extra-embryonic membrane
Blocking step
Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 23°C

Abcam user community

Verified customer

Submitted Jul 15 2014

Question
Answer

Thank you for contacting us.

I have attached the results of the sequence alignment between mouse and human angiopoietin 1.

The link to our IHC-P protocol is as follows:
https://www.abcam.com/index.html?pageconfig=resource&rid=11384

The file is also attached for your convenience.

I hope this information is helpful to you. Please do not hesitate to contact us if you need any more advice or information.

Use our products? Submit an Abreview. Earn rewards!
https://www.abcam.com/abreviews

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Answer

Thanks again for the additional data. I think there are a few confounding factors here that make the western blot data difficult to interpret confidentally: 1. I agree with you that the intense 40kDa band is likelyb-actin in both blots, but we can not be certain. 2. There also may be bleed-through from the first anti-angiopoietin blot to the second angiopoietin blot. 3. Pig angtiopoietin-1 has technically not been tested previously with either ab8451 or ab95230, so the specificity of these antibodies with respect to pig samples is not known. 4. The expression pattern ofangtiopoietin-1 in pig kidney tissue, including any alternate forms or glycosylated forms, are not known, at least based on my brief literature search. You may consider treating your samples with or without PNGase-F, an endoglycosylase which would remove complex oligosaccharides from N-linked glycoproteins. If treated samples show a band with a smaller molecular weight, this suggests the detected protein was gylcosylated and likely angtiopoietin-1. I look forward to hearing if these suggestions prove useful.

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Answer

Thanks for the additional information. A couple more questions... 1. Are one or more of the samples from the blot considered a negative control, in that it is known to not express Angiopoietin 1? 2. After stripping but before reprobing, was it determined that there was no residual ECL signal remaining from the previous blot? Thanks again!

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Answer

Thanks for providing the western blot images as well as the protocol and sample preparation details. I just have a few additional questions: 1. Was any other antibody usedsuccessfully in western blotting with these same samples? 2. How much protein was loaded per lane? 3. Angiopoietin may be glycosylated and may contain disulfide bonds. This may account for bands at higher molecular weights than expected. Was reducing agent used in sample prep? 4. Are the samples in each lane of the both blots the same, ie. is the lane 1 sample in the ab8451 blot the same sample as that run in lane 1 on the ab95230 blot? Thanks in advance for the additional information.

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