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: IHC-P, WB, IHC-Frmore details
  • Species reactivity
    Reacts with: Mouse, Human, Pig
  • Immunogen

    Synthetic peptide:

    CNQRRNPENGGRRYNRIQHGQ

    , corresponding to N terminal amino acids 21/40 of Mouse Angiopoietin 1. (Peptide available as ab9076).

  • General notes


    Angiopoeitin-1 (Ang-1) and Antiopoietin-2 (Ang2) are important for development of the endothelium, by regulating tyrosine phosphorylation of the membrane receptor Tie-2/Tek. Ang-1 binding to Tie-2/Tek causes phosphorylation of the receptor. Ang-2 competes for this binding, and thus blocks receptor phosphorylation. Ang-1 has potential fibrinogen-like domain at the carboxy terminus and coiled-coil regions in the amino terminus. Ang-1 is prominently expressed in the myocardium of atrium and ventricle, mesenchymal and smooth muscle cells surrounding most blood vessels, and lung. In the adult, ANG-1 is also expressed in the heart and liver. To our knowledge, this is the first time that an Ang antibody recognises a band above 55 kD. This antibody (and the ang-2 antibody, ab8452) give a band at 75 kD which resembles the original size, because these are soluble highly glycosylated proteins, which should run higher than the calculated molecular weight of 55 kD.

Properties

  • Form
    Liquid
  • Storage instructions
    Shipped at 4°C. Upon delivery aliquot and store at -20°C or -80°C. Avoid repeated freeze / thaw cycles.
  • Storage buffer
    pH: 7.20
    Preservative: 0.01% Sodium azide
    Constituents: 0.42% Potassium phosphate, 0.87% Sodium chloride
  • Purity
    Whole antiserum
  • Primary antibody notes
    Angiopoeitin-1 (Ang-1) and Antiopoietin-2 (Ang2) are important for development of the endothelium, by regulating tyrosine phosphorylation of the membrane receptor Tie-2/Tek. Ang-1 binding to Tie-2/Tek causes phosphorylation of the receptor. Ang-2 competes for this binding, and thus blocks receptor phosphorylation. Ang-1 has potential fibrinogen-like domain at the carboxy terminus and coiled-coil regions in the amino terminus. Ang-1 is prominently expressed in the myocardium of atrium and ventricle, mesenchymal and smooth muscle cells surrounding most blood vessels, and lung. In the adult, ANG-1 is also expressed in the heart and liver. To our knowledge, this is the first time that an Ang antibody recognises a band above 55 kD. This antibody (and the ang-2 antibody, ab8452) give a band at 75 kD which resembles the original size, because these are soluble highly glycosylated proteins, which should run higher than the calculated molecular weight of 55 kD.
  • Clonality
    Polyclonal
  • Isotype
    IgG
  • Research areas

Applications

Our Abpromise guarantee covers the use of ab8451 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
IHC-P Use at an assay dependent concentration.
WB 1/500. Detects a band of approximately 57 kDa. Using this antiserum with cell supernatants may result in high background due to other components in the serum. This can be alleviated by immunoprecipitating the antibody:antigen complex before detection, or precipitation using soluble Tie2. These methods result in a clean, strong signal. Both Ang-1 and Ang-2 proteins have predicted MW of 57 kDa and appear on blots close to predicted MW. Additional bands may be seen at approx. 75 kDa which represent highly glycosylated forms of the protein.
IHC-Fr Use at an assay dependent concentration.

Target

  • Function
    Binds and activates TIE2 receptor by inducing its tyrosine phosphorylation. Implicated in endothelial developmental processes later and distinct from that of VEGF. Appears to play a crucial role in mediating reciprocal interactions between the endothelium and surrounding matrix and mesenchyme. Mediates blood vessel maturation/stability. It may play an important role in the heart early development.
  • Sequence similarities
    Contains 1 fibrinogen C-terminal domain.
  • Post-translational
    modifications
    Glycosylated.
  • Cellular localization
    Secreted.
  • Information by UniProt
  • Database links
  • Alternative names
    • AGP 1 antibody
    • AGP1 antibody
    • AGPT antibody
    • ANG 1 antibody
    • ANG antibody
    • ANG-1 antibody
    • ANG1 antibody
    • Angiopoietin-1 antibody
    • Angiopoietin1 antibody
    • ANGP1_HUMAN antibody
    • ANGPT 1 antibody
    • Angpt1 antibody
    • KIAA0003 antibody
    • OTTHUMP00000227592 antibody
    • OTTHUMP00000227599 antibody
    • OTTHUMP00000227600 antibody
    see all

Images

  • Supernatants of mouse-angiopoietin-expressing endothelial cells.

    Lane 1 - wt endothelial cell
    Lane 2 - mouse Ang-1 (clone 1-8) expressing cells
    Lane 3 - mouse Ang-1 (clone 1-15) expressing cells
    Lane 4 - mouse Ang-2 (clone 2-9) expressing cells

    Supernatants of mouse-angiopoietin-expressing endothelial cells.

    Lane 1 - wt endothelial cell
    Lane 2 - mouse Ang-1 (clone 1-8) expressing cells
    Lane 3 - mouse Ang-1 (clone 1-15) expressing cells
    Lane 4 - mouse Ang-2 (clone 2-9) expressing cells

  • ab8451 was diluted 1:500 to detect Angiopoietin 1 in human lung tissue. Tissue was formalin fixed and paraffin embedded. No pre-treatment of sample was required. The image shows the localization of antibody as the precipitated red signal, with a hematoxylin purple nuclear counter stain.

References

This product has been referenced in:
  • Smeda M  et al. Dual antiplatelet therapy with clopidogrel and aspirin increases mortality in 4T1 metastatic breast cancer-bearing mice by inducing vascular mimicry in primary tumour. Oncotarget 9:17810-17824 (2018). Read more (PubMed: 29707148) »
  • Magkouta S  et al. Targeting Tie-2/angiopoietin axis in experimental mesothelioma confers differential responses and raises predictive implications. Oncotarget 9:21783-21796 (2018). Read more (PubMed: 29774102) »
See all 28 Publications for this product

Customer reviews and Q&As

1-10 of 22 Abreviews or Q&A

Answer

Thank you for contacting us.
I changed the review to editing status. You can now change the Abreview by correcting the details.
Please also confirm that the secondary antibody was polyclonal or monoclonal?
ab8451 is a rabbit antibody so secondary antibody can't rabbit.
Please check these and update the Abreview. Thanks!

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Answer

Vielen Dank für diese weiteren Informationen.
Es tut mir Leid, dass Sie Probleme mit diesem Antikörper hatten. Wie am Telefon besprochen, habe ich eine kostenlose Ersatzlieferung für Sie in Auftrag gegeben. Sie hat die Referenznummer 123456 und sollte morgen bei Ihnen ankommen.
Ich kann die Blots bezüglich der KO Mäuse gut gebrauchen: Das gibt mir den Rückhalt gegenüber dem Labor nicht nur das betroffene Lot zurück zurufen, sondern dass die Lots in Produktion noch einmal genauer angeschaut werden!
Ich wünsche Ihnen viel Erfolg mit Ihren Projekten. Bitte melden Sie sich wieder bei uns, falls es weitere Fragen oder Probleme gibt.

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Question
Answer


Es tut mir Leid, dass Sie Probleme mit diesen Antikörpern hatten. Wie am Telefon besprochen habe ich eine kostenlose Ersatzlieferung für Sie in Auftrag gegeben. Sie hat die Referenznummer 123456 und sollte allerdings erst am 5 September bei Ihnen ankommen ( wir müssen noch das alternative Lot für den Angiopoietin 1 aus Japan einfliegen lassen).

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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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Question

Here is my completed questionnaire I think the only omission is the date we ordered the antibody but I think it must be almost 6 months ago.

We have intermittently tried to get this antibody to work and this is our last and can I add desperate attempt.

I will have to send the images separately as they keep bouncing back

Thank you for your help

Regards

Order Details
Antibody code: AB 8451
Lot number: GR 24123-2
Purchase order number
or preferably Abcam order number:
General Information
Antibody storage conditions (temperature/reconstitution etc)
-20
Description of the problem (high background, low signal, non-specific satining etc.)
No signal
Or nuclear staining
Or high background
Sample (Species/Tissue/Cell Type/Cell Line etc.)
Human lung tissue explanted at time of transplant.
Fixation of sample (Ethanol/Methanol/Acetone/Paraformaldehyde/Other/Duration etc.)
Formalin fixative unknown duration
Antigen retrieval (Enzymatic method, Heat mediated technique etc.)
Trypsin digestion
Citrate pH 6 and EDTA pH 8
At 60o
Or MW
Or Pressure
Permeabilization step
Tween 20 in TBS 10 mins pre primary
Blocking conditions (Buffer/time period, Blocking agent etc.)
Endogenous peroxidase blocked in Methanolic H2O2 for 30 mins pre retrieval.
Primary Antibody (Manufacturer/Species/Diluent/Dilution/Incubation time, Wash step)
1:250 in 3% FBS 1 hour at RT or 4o overnight
Secondary Antibody (Manufacturer/Species/Diluent/Dilution/Incubation time, Wash step)
Dako Envision anti rabbit kit RTU
Detection method
Dako DAB supplied with Envision Kit
Positive and negative controls used (please specify)
Placenta
Asthmatic lung
Normal Rabbit igG
Optimization attempts (problem solving)
How many times have you tried the IHC?
lots
Have you run a "No Primary" control?
Yes No
Do you obtain the same results every time?
Yes No
What steps have you altered?
Antigen retrieval
Additional Notes
I realize that fixation is important but unfortunately we have no control over this stage. Our blocks are on loan from a routine cellular pathology lab. They also have been archived over a period of 12 years.

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Answer

Thank you for taking the time to complete our questionnaire and get back to me.

The details you have kindly provided will enable us to investigate this case for you and this is also helpful in our records for monitoring of quality.

Reviewing this case, I would like to offer some suggestions to help optimise the results from ab8451. I would also appreciate if you can confirm some further details:

1. Please provide the order number and date of purchase.

2. In order to help me assess the results:

What conditions are being using when there is no signal? What was changed afterwards to improve try and improve the results?

What conditions are being used when there is nuclear staining? What was changed afterwards to improve try and improve the results?

What conditions are being used when there is high background? What was changed afterwards to improve try and improve the results?

3. It would be beneficial if you are able to provdie some images? I am sorry you have had difficulty submitting these. It may be that the file size is too large, you could try compressing and sending them as a PDF to see if this works. If you are still having difficulty sending these, please do not hesitate to try sending them to my personal email address kate.hayes@abcam.com.

Please send any text reply by direct reply to this email address (technical@abcam.com) and images to my personal address.

4. I can suggest to try different times for antigen retreival as this can sometimes require some optimization if this has not already been tried.

5. Is the anti rabbit kit and detection kit working well with other primary antibodies?

6. The samples may be varying in quality, especially if they have been collected over so many years at the hospital. Is there any opportunity to try newer samples prepared over the last few weeks?

I hope this information is helpful, thank you for your cooperation. Should the suggestions not improve the results, please do not hesitate to contact me again with the further requested details.

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Answer

Thank you for taking the time to contact us. I am sorry to hear you have had difficulty obtaining satisfactory results from this antibody and we are happy to help.

I would like to reassure you that this antibody is tested and covered by our 6 month guarantee for IHC-P and human samples. In the event that a product is not functioning in the tested applications and species cited on the product data sheet (and the problem has been reported within 6 months of purchase), we will be pleased to provide a credit note, free of charge replacement or refund.

I would like to investigate this particular case further for you, and also obtain more information for our quality monitoring records. In order to proceed with this, I have enclosed a technical questionnaire below. I would appreciate if you could complete this. It will help you put the information we require together very easily. Once we have this information, I hope to be able to provide some suggestions as requested.

I would appreciate if you could also provide an image which would help us to assess the results.

Thank you for your time and cooperation. We look forward to receiving the completed questionaire.
Order Details
Antibody code:
Lot number:
Purchase order number
or preferably Abcam order number:
General Information
Antibody storage conditions (temperature/reconstitution etc)
Description of the problem (high background, low signal, non-specific satining etc.)
Sample (Species/Tissue/Cell Type/Cell Line etc.)
Fixation of sample (Ethanol/Methanol/Acetone/Paraformaldehyde/Other/Duration etc.)
Antigen retrieval (Enzymatic method, Heat mediated technique etc.)
Permeabilization step
Blocking conditions (Buffer/time period, Blocking agent etc.)
Primary Antibody (Manufacturer/Species/Diluent/Dilution/Incubation time, Wash step)
Secondary Antibody (Manufacturer/Species/Diluent/Dilution/Incubation time, Wash step)
Detection method
Positive and negative controls used (please specify)
Optimization attempts (problem solving)
How many times have you tried the IHC?
Have you run a "No Primary" control?
Yes No
Do you obtain the same results every time?
Yes No
What steps have you altered?
Additional Notes
We would appreciate if you are also able to provide and image which woudl help us to assess the results

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Answer

Thank you for getting back to us. Unfortunately, xxx is away from the office today so I will deal with this enquiry.

I think what xxxx was suggesting was to perform a "no primary" control, in order to determine if the secondary antibody was making any contribution to the non-specificity observed, as well as a sample known no to express Angiopoietin 1. Unfortunately, we do not have this to provide.I would suggest your customer perform this as well as the additional suggestions Kate made:

1. I can suggest to try incubate overnight 4oC which can often provide more efficient and specific staining. I can also recommend to try a lower dilution, 1:2000 to reduce the non specific staining

2. Could you confirm the wash steps? I can suggest to wash 4 times for 5 minutes in PBS containing 0.2% Tween at the appropriate wash steps if this has not already been tried. Also, changing from PBS to TBS can provide a more stringent wash.

Additionally, could you confirm the following:

1. Could you confirm if the current vial of secondary antibody is working well with other primary antibodies?

2. Could you confirm if a loading control staining has been included to help asses the quality of the samples?

Many thanks for your continued assistance.

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Question

Product code: 8451
Lot number: GR24123
Inquiry: Multi bands The data was shown multi-bands in the same sample. I would like to know what band indicates angiopoietin-1 protein. Please advice and comment about this data.

1) Abcam product code
ab8451

2) Abcam order reference number or product batch number
GR24123

storage temperature of antibody -80oC

3) Description of the problem
Multi bands

4) Sample preparation:
Type of sample and species(whole cell lysates, fraction, recombinant protein and Human,
mouse; mouse brain

Lysis buffer RIPA buffer (Sigma)

Protease inhibitors: Protease inhibitor cocktail

Phosphatase inhibitors : no

Reducing agent: DTT

Boiling for 5 min? 10min at 90oC
Protein loaded ug/lane or cells/lane : 50 ug/lane

Positive control : no
Negative control : no

5) Percentage of gel : 4-12% Bis-Tris gels

Type of membrane : PVDF membrane
Protein transfer verified : Ponceau S

Blocking agent and concentration : 5% BSA in TBST
Blocking time : 1 hour
Blocking temperature : room temperature (RT)

6) Primary antibody (If more than one was used, describe in ¡°additional notes¡±) :
Concentration or dilution : 1:1000
Diluent buffer : 5% BSA in TBST
Incubation time : 1 hour
Incubation temperature: RT

7) Secondary antibody:
Species: goat anti-rabbit IgG-HRP (invitrogen-g-21234)
Isotype: IgG
Reacts against: rabbit
Concentration or dilution : 1:3000
Diluent buffer : 5% BSA
Incubation time : 30 min
Incubation temperature: RT
Fluorochrome or enzyme conjugate: HRP

8) Washing after primary and secondary antibodies:
Buffer : TBST
Number of washes : 3 times

9)Detection method ECL

10) How many times have you run this staining? : 4 times
-Do you obtain the same results every time? : yes
-Have you run a No Primary control? (yes/no) : no

-What steps have you altered to try and optimize the use of this antibody?

Document attachment: Attaching images of your blot is strongly recommended and can greatly speed up our investigation of your problem.

Read More
Answer

Thank you for taking the time to complete our questionnaire and contact us. I am sorry to hear you have had difficulty obtaining satisfactory results from this antibody.

The details you have kindly provided will enable us to investigate this case for you and this is also helpful in our records for monitoring of quality.

I would like to reassure you that ab8451 is tested and covered by our 6 month guarantee for use inWB and human and mouse samples. In the event that a product is not functioning in the applications cited on the product data sheet, we will be pleased to provide a credit note or free of charge replacement.

Regarding the results, there should be a single band at 57 kDa (sometimes there is a higher band at around 75 kDa which indicates glycosylated protein, but your results do not show this). Reviewing this case, I would like to offer some suggestions to help optimize the results. I would also appreciate if you can confirm some further details to assist in my investigation of this case.

1. I can suggest it would be beneficial to consider including an endogenous negative control samples. Could you confirm if this has been tried?

2. Could you confirm if the current vial of secondary antibody is working well with other primary antibodies?

3. I can suggest to try incubate overnight 4oC which can often provide more efficient and specific staining. I can also recommend to try a lower dilution, 1:2000 to reducethe non specific staining

4. Could you confirm the wash steps? I can suggest to wash 4 times for 5 minutes in PBS containing 0.2% Tween at the appropriate wash steps if this has not already been tried. Also, changing from PBS to TBS can provide a more stringent wash.

5. Could you confirm if a loading control staininghas been includedto help asses the quality of the samples?

I hope this information is helpful, thank you for your cooperation. Should the suggestions not improve the results, please do not hesitate to contact me again with the further requested details.

Read More

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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1-10 of 22 Abreviews or Q&A

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