Overview

Properties

Applications

Our Abpromise guarantee covers the use of ab9570 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
Sandwich ELISA Use at an assay dependent concentration. Can be paired for Sandwich ELISA with Rabbit polyclonal to VEGFA (Biotin) (ab83132).

To detect hVEGF by sandwich ELISA (using 100 µl/well antibody solution) a concentration of 0.5 - 2.0 µg/ml of this antibody as Detection is required. This antigen affinity purified antibody, paired with the above recommended pair, allows the detection of at least 0.2 - 0.4 ng/well of recombinant hVEGF (ab9571).

ELISA Use at an assay dependent concentration. To detect hVEGF by direct ELISA (using 100µl/well antibody solution) a concentration of at least 0.5µg/ml of this antibody is required. This antigen affinity purified antibody, in conjunction with compatible secondary reagents, allows the detection of 0.2 - 0.4 ng/well of recombinant hVEGF.
Neutralising Use at an assay dependent concentration. To yield one-half maximal inhibition [ND50] of the biological activity of hVEGF (10.0 ng/ml), a concentration of 0.05 - 0.1 µg/ml of this antibody is required.
WB Use a concentration of 0.1 - 0.2 µg/ml.
IHC-P Use a concentration of 2.5 - 5.5 µg/ml. Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.

Target

  • Function
    Growth factor active in angiogenesis, vasculogenesis and endothelial cell growth. Induces endothelial cell proliferation, promotes cell migration, inhibits apoptosis and induces permeabilization of blood vessels. Binds to the FLT1/VEGFR1 and KDR/VEGFR2 receptors, heparan sulfate and heparin. NRP1/Neuropilin-1 binds isoforms VEGF-165 and VEGF-145. Isoform VEGF165B binds to KDR but does not activate downstream signaling pathways, does not activate angiogenesis and inhibits tumor growth.
  • Tissue specificity
    Isoform VEGF189, isoform VEGF165 and isoform VEGF121 are widely expressed. Isoform VEGF206 and isoform VEGF145 are not widely expressed.
  • Involvement in disease
    Defects in VEGFA are a cause of susceptibility to microvascular complications of diabetes type 1 (MVCD1) [MIM:603933]. These are pathological conditions that develop in numerous tissues and organs as a consequence of diabetes mellitus. They include diabetic retinopathy, diabetic nephropathy leading to end-stage renal disease, and diabetic neuropathy. Diabetic retinopathy remains the major cause of new-onset blindness among diabetic adults. It is characterized by vascular permeability and increased tissue ischemia and angiogenesis.
  • Sequence similarities
    Belongs to the PDGF/VEGF growth factor family.
  • Cellular localization
    Secreted. VEGF121 is acidic and freely secreted. VEGF165 is more basic, has heparin-binding properties and, although a signicant proportion remains cell-associated, most is freely secreted. VEGF189 is very basic, it is cell-associated after secretion and is bound avidly by heparin and the extracellular matrix, although it may be released as a soluble form by heparin, heparinase or plasmin.
  • Information by UniProt
  • Database links
  • Alternative names
    • Folliculostellate cell-derived growth factor antibody
    • Glioma-derived endothelial cell mitogen antibody
    • MGC70609 antibody
    • MVCD1 antibody
    • Vascular endothelial growth factor A antibody
    • vascular endothelial growth factor A121 antibody
    • vascular endothelial growth factor A165 antibody
    • vascular endothelial growth factor antibody
    • Vascular permeability factor antibody
    • VEGF A antibody
    • Vegf antibody
    • VEGF-A antibody
    • VEGF120 antibody
    • Vegfa antibody
    • VEGFA_HUMAN antibody
    • VPF antibody
    see all

Images

  • ab9570 staining VEGF in human breast invasive ductal carcinoma section by Immunohistochemistry (Formalin/PFA fixed paraffin-embedded sections). Tissue underwent heat mediated antigen retrieval in sodium citrate buffer (pH 6.0). The primary antibody was used at 2.5 ug/ml and incubated with sample at 4°C overnight. A HRP-labeled polymer detection system was used with a DAB chromogen.

  • Lanes 2-12 : Anti-VEGFA antibody (ab9570) at 0.2 µg/ml

    Lane 1 : MultiMark MultiColor Standard
    Lane 2 : Recombinant Human VEGF at 0.25 µg
    Lane 3 : Recombinant Human VEGF at 0.125 µg
    Lane 4 : Recombinant Human VEGF at 0.0625 µg
    Lane 5 : Recombinant Human VEGF at 0.03125 µg
    Lane 6 : Recombinant Human VEGF at 0.015625 µg
    Lane 7 : Recombinant Human VEGF at 0.0078 µg
    Lane 8 : Recombinant Human VEGF at 0.0039 µg
    Lane 9 : Recombinant Human VEGF at 0.00195 µg
    Lane 10 : Recombinant Human VEGF at 0.000975 µg
    Lane 11 : Recombinant Human VEGF at 0.0004875 µg
    Lane 12 : Recombinant Human VEGF at 0.00024 µg

    Performed under non-reducing conditions.

    Observed band size: 38.2 kDa
    why is the actual band size different from the predicted?

  • Detected human VEGF in a sandwich ELISA within the range 16-1000 pg/ml
    - 0.5 µg/ml of capture antibody (ab9570)
    - 0.25 µg/ml of detection antibody (ab83132)
  • Lane 1 : Molecular weight ladder
    Lanes 2-12 : Anti-VEGFA antibody (ab9570)

    Lane 2 : Recombinant human VEGFA at 0.25 µg
    Lane 3 : Recombinant human VEGFA at 0.125 µg
    Lane 4 : Recombinant human VEGFA at 0.0625 µg
    Lane 5 : Recombinant human VEGFA at 0.03125 µg
    Lane 6 : Recombinant human VEGFA at 0.015625 µg
    Lane 7 : Recombinant human VEGFA at 0.0078 µg
    Lane 8 : Recombinant human VEGFA at 0.0039 µg
    Lane 9 : Recombinant human VEGFA at 0.00195 µg
    Lane 10 : Recombinant human VEGFA at 0.000975 µg
    Lane 11 : Recombinant human VEGFA at 0.0004875 µg
    Lane 12 : Recombinant human VEGFA at 0.00024 µg

    Performed under reducing conditions.

References

This product has been referenced in:
  • Xie M  et al. MicroRNA-1 acts as a tumor suppressor microRNA by inhibiting angiogenesis-related growth factors in human gastric cancer. Gastric Cancer 21:41-54 (2018). WB . Read more (PubMed: 28493075) »
  • Siddharth S  et al. The soluble nectin-4 ecto-domain promotes breast cancer induced angiogenesis via endothelial Integrin-ß4. Int J Biochem Cell Biol 102:151-160 (2018). Read more (PubMed: 30056265) »
See all 10 Publications for this product

Customer reviews and Q&As

1-10 of 13 Abreviews or Q&A

Question

Hi Carolyn,

I wasn't able to find the attached report, but it sounds like what we were expecting. If you could send a small sample of protein for 1 Western blot to confirm that I can detect the same fragments (monomer and perhaps a little residual dimer) that would be great. This way I can compare it with the VEGF we have been using to see if there is a difference. Do you know what reducing conditions Abcam uses? I use 20mM DTT.

Thanks,
Daniel



On Fri, Jun 1, 2012 at 1:27 PM, <mailto:us.technical@abcam.com> wrote:














https://www.abcam.com/index.html?utm_campaign=CRM&utm_source=Abcam.CRM&utm_medium=Email&utm_term=Header








Dear Daniel



Thank you for this information.

I have had the lab do some further investigation with this antibody.We found one that was run reduced rather than the unreduced one I previously sent. We have spoken with the antibody lab to confirm, but our antibody detected the reduced protein at a MW of just below 20kDa with a slight band of residual unreduced protein at 40kDa. I have attached the report for your review. Based on this information and some discussion with our labs, we do not believe the issue is with the antibody. It seems that our antibody works fine for detecting reduced and unreduced VEGF protein.

Would you be interested in testing our VEGF protein?

Help us improve our service.
https://www.abcam.com/index.html?pageConfig=technicalSurvey&intCCEID=3816557


Best regards,
Carolyn

Carolyn Miazga, PhD
Scientific Support Supervisor
Abcam Inc.
https://www.abcam.com


Your original inquiry to Abcam:


Hi Carolyn,



The samples sit in DTT for probably a few minutes while I finish mixing all of the samples, then I heat them at 95 C for 5 minutes after which they probably sit a few more minutes while I finish preparing the gel. So it is roughly 10-12 minutes from mixing to loading the gel.



Daniel


On Thu, May 31, 2012 at 3:14 PM, <mailto:us.technical@abcam.com> wrote:














https://www.abcam.com/index.html?utm_campaign=CRM&utm_source=Abcam.CRM&utm_medium=Email&utm_term=Header








Dear Daniel



Thank you for your patience.

The lab had an additional question: how longare youletting the protein + DTT sit before running the Western blot assay?

Help us improve our service.
https://www.abcam.com/index.html?pageConfig=technicalSurvey&intCCEID=3813097


Best regards,
Carolyn

Carolyn Miazga, PhD
Scientific Support Supervisor
Abcam Inc.
https://www.abcam.com


Your original inquiry to Abcam:


Hi Carolyn,



I have attached a picture of my Western blot. Lane 1 is the standard, lane 2 the VEGF and lane 3 a more dilute VEGF. I am not seeing the dimer at all(I reduced with 20mM DTT). But the band around 10kDa is pretty strong. The standard has the following MWs from bottom to top: 6.4, 15.0, 20.6, 27.6, 41.1, 55.8, 102 or 195 kDa (I think one of the top two bands is not showing up well). The residual dimer appears at just below 41.1kDa, which makes sense, but the low MW fragment is appearing between 6.4kDa and 15.0kDa. The only thing I can think of is that this is actually the dimer, but for some reason is showing up really low.



Thanks,
Daniel




On Thu, May 24, 2012 at 1:02 PM, <mailto:us.technical@abcam.com> wrote:














https://www.abcam.com/index.html?utm_campaign=CRM&utm_source=Abcam.CRM&utm_medium=Email&utm_term=Header








Dear Daniel



Thank you for contacting Abcam regarding ab9570.

I am sorry to hear you have been obtaining some confusing results with this antibody. I have checked with the lab and the same isoform of VEGF that you have purchased was also used as the immunogen to generate this antibody.

I have confirmed with the laboratory, that at higher concentrations of VEGF, the dimer, monomer and a 10kDa band is detected by this antibody, but those bands disappear at lower concentrations of protein. We are unsure as to the identity of the 10kDa band. Please see our datasheet for the WB image. Would you also send us an image of your blot?

Finally, I have also confirmed that we are able to obtain the recombinant protein that was used as the immunogen. If you are interested in this product, then Iwill request it be added to our catalog for purchase.

I hope this information is helpful. Please do not hesitate to contact us if you have any additional questions.

Help us improve our service.
https://www.abcam.com/index.html?pageConfig=technicalSurvey&intCCEID=3795097


Best regards,
Carolyn

Carolyn Miazga, PhD
Scientific Support Supervisor
Abcam Inc.
https://www.abcam.com


Your original inquiry to Abcam:


Name: Daniel Alt

Product code: 9570

Lot number: GR9232-4



Inquiry: Hello, I am using ab9570 anti-rhVEGF antibody to detect R&D System's rhVEGF (293-ve). I get detection of a small amount of unreduced dimer of VEGF at the appropriate molecular weight (˜40kDa), but the monomer is not appearing at ˜20kDa. Rather, I am getting a strong signal at ˜10kDa. I have contacted R&D about this and after checking a few other things, they said to check what VEGF the antibody was raised against because it might not recognize their particular VEGF monomer. Although, I am still confused why I get such a high intensity signal at 10kDa, I was wondering what VEGF the ab9570 antibody was raised against. Was this a commercially available VEGF from Abcam? Does Abcam have a commercially available anti-VEGF antibody raised against R&D Systems VEGF? Thanks.








Abcam Customer Services and Scientific Support Team
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Abcam Customer Services and Scientific Support Team
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[CCE3813097]






















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Abcam Customer Services and Scientific Support Team
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Read More
Answer

Thank you for your patience.


I just wanted to let you know that the protein has been ordered and you will be receiving this free of charge during the first week of July.

Please do not hesitate to contact me if you have any additional questions.

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Answer

It is always a pleasure to help.

Please do not hesitate to contact us should you have any questions in the future.

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Question
Answer

Thank you for contacting us.

This product is sold in a 100ug size at a concentration of 0.5mg/ml. Therefore each vial should contain 200ul.

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

Thank you for this information.


I have had the lab do some further investigation with this antibody.We found one that was run reduced rather than the unreduced one I previously sent. We have spoken with the antibody lab to confirm, but our antibody detected the reduced protein at a MW of just below 20kDa with a slight band of residual unreduced protein at 40kDa. I have attached the report for your review. Based on this information and some discussion with our labs, we do not believe the issue is with the antibody. It seems that our antibody works fine for detecting reduced and unreduced VEGF protein.


Would you be interested in trying our VEGF protein?

Read More

Answer

Thank you for contacting Abcam regarding ab9570.


I am sorry to hear you have been obtaining some confusing results with this antibody. I have checked with the lab and the same isoform of VEGF that you have purchased was also used as the immunogen to generate this antibody.


I have confirmed with the laboratory, that at higher concentrations of VEGF, the dimer, monomer and a 10kDa band is detected by this antibody, but those bands disappear at lower concentrations of protein. We are unsure as to the identity of the 10kDa band. Please see our datasheet for the WB image. Would you also send us an image of your blot?


Finally, I have also confirmed that we are able to obtain the recombinant protein that was used as the immunogen. If you are interested in this product, then I will request it be added to our catalog for purchase.


I hope this information is helpful. Please do not hesitate to contact us if you have any additional questions.

Read More

Answer

Thank you for contacting us.

For the proteins, I would suggest using ab61861 as this protein is arecombinant Human VEGF 165A protein (double, non-glycosylated, polypeptide chain containing 165 amino acids, MW 38kDa, expressed in E.coli).

In terms of the most suitable antibodies, ab9570 which is reactive with VEGF 165and 121, would be the best choice. It was raised against highly pure (>98%) recombinant hVEGF 165 (human Vascular Endothelial Cell Growth Factor), however, the epitope has not been mapped.

As for ab52917 and ab99511, I willcontact the lab to obtain more information about the immunogen region. As soon as I have more details, I will let you know.

What kind of conformation studies are you planning to do? As far as I can tell such application has not been tried with neither these protein nor antibodies.

I hope this information is already of some help to you.
In the meantime, please do not hesitate to contact us if you need any more advice or information.

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Question
Answer

Thank you for contacting us.

Thefollowing CROSS REACTIVITY has been tested with the antibodies ab9570 and ab83132:

When prepared at 50ng/ml the following antigens exhibited 100% cross reactivity:

Human VEGF121
Murine VEGF
Rat VEGF

When prepared at 50ng/ml the following antigens did NOT exhibit significant cross reactivity:

Human EGF, EG-VEGF, FGF-16, GM-CSF, PDGF-AA, PDGF-AB, PDGF-BB, PDGF-CC, RANTES, SCF, VEGF-B, VEGF-C, VEGF-D
Murine EGF, GM-CSF, PDGF-AA, PDGF-BB, SCF
Rat EGF, SCF

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

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Question
Answer

Thank you for contacting us.



The WB protocol is as follows:

1. After the protein has been transferred to the nitrocellulose membrane, incubate it in a blocking solution containing 3% Nonfat Dry Milk. The membrane should block for 30 minutes at room temperature on a shaker.

2. Wash the membrane three times in PBST (PBS with 0.1% TWEEN-20) in a clean tray on a shaker. Each of the three washes should last for 15 minutes.

3.Dilute the probing antibody to the desired concentration in PBST; final volume= 50ml. Incubate the membrane on the shaker in a clean tray containing the antibody solution for one hour at room temperature. The actual time for this incubation may be longer depending on the affinity/avidity of the probing antibody (up to four hours).

4. Wash the membrane three more times following the same wash procedure in step #2.

5. The secondary antibody (species-anti-probing species-Alk Phos) should be diluted 1:2,000 in PBST. Incubate the membrane in a clean tray containing the diluted secondary antibody on the shaker for one hour at room temperature.

6.Wash the membrane three times following the procedure in step #2.

7.DEVELOPMENT:
For development use alkaline phosphatase conjugated antibodies. Let the membrane develop until the bands are clearly visible. Stop the color development reaction by placing the membrane in a tray containing ddH2O for at least 10 minutes.
--------------------------------------------------------------------------------


The direct ELISA protocol is as follows:

RECOMMENDED SOLUTIONS
All solutions should be at ambient temperature prior to use.
PBS: Dilute 10xPBS to 1xPBS, pH 7.20 in sterile water.
Wash Buffer: 0.05% Tween-20 in PBS
Block Buffer: 1% BSA in PBS*
Diluent: 0.05% Tween-20, 0.1% BSA in PBS*
Substrate Solution: ABTS Liquid Substrate Solution
*Sterile filter and store at 4°C for up to 1 week.

PLATE PREPARATION

Standard/Sample: Serial dilute standard from 0.1μg/ml to zero in PBS. Add 100μl of standard or sampleto each well in triplicate. Incubate at room temperature overnight.
Aspirate the wells to remove liquid and wash plates 4 times. Each wash consists of adding 300μl washbuffer per well, followed by aspiration. After the last wash invert plate to remove residual buffer andblot on paper towel.
Add 300μl blocking buffer to each well. Incubate 2 hour at R.T.
Aspirate and wash plate 4 times (as in step 2).



ELISA PROTOCOL
Detection: Wash plate four times. Dilute detection antibody (biotinylated) in diluent to a concentration of1 μg/ml. Immediately add 100μl per well. Incubate at room temperature for 2 hours.

Avidin-HRP Conjugate:Aspirate and wash plate 4 times. Dilute Avidin-HRP conjugate 1:2000 in diluent.
Add 100μl per well. Incubate 30 min at room temperature.

Substrate:Aspirate and wash plate 4 times. Add 100μl of substrate solution to each well. Incubate at room temperature for color development. Monitor color development with an ELISA plate reader at 405 nm with wavelength correction set at 650 nm.

NOTE: Reliable standard curves are obtained when O.D. readings do not exceed 0.2 units for the zero standard concentrations, or 1.2 units for the highest standard concentration. The plate should be monitored at 5 minute intervals until desired O.D. readings are obtained. The typical range is 5-40 minutes. O.D. readings may vary.

Assay sensitivity may be increased with additional washings.
------------------------------------------------------------------------------

The sandwich ELISA protocol is as follows:

RECOMMENDED MATERIALS
ELISA microplates (Nunc MaxiSorp Prod. # 439454, or Corning Prod. # 3590);
Tween-20
BSA
Avidin-HRP conjugate
ABTS Liquid Substrate Solution
Dulbecco’s PBS [10x]

RECOMMENDED SOLUTIONS
All solutions should be at ambient temperature prior to use.
PBS: Dilute 10xPBS to 1xPBS, pH 7.20 in sterile water.
Wash Buffer: 0.05% Tween-20 in PBS
Block Buffer: 1% BSA in PBS*
Diluent: 0.05% Tween-20, 0.1% BSA in PBS*
*Sterile filter and store at 4°C for up to 1 week.

PLATE PREPARATION

Dilute capture antibody (polyclonal) with PBS to a concentration of 1μg/ml. Immediately, add 100μl to eachELISA plate well. Seal the plate and incubate overnight at room temperature. (Monoclonal Antibody – at least 2 μg/ml).
Aspirate the wells to remove liquid and wash plates 4 times. Each wash consists of adding 300μl wash bufferper well, followed by aspiration. After the last wash invert plate to remove residual buffer and blot onpaper towel.
Add 300μl blocking buffer to each well. Incubate 1 hour at R.T.
Aspirate and wash plate 4 times (as in step 2).




ELISA PROTOCOL
Standard/Sample: Serial dilute standard from 0.01μg/ml to zero in diluent. Add 100μl of standard or sample to each well in triplicate. Incubate at room temperature for at least 2 hours.

Detection: Wash plate four times. Dilute detection antibody (biotinylated) in diluent to a concentration of0.5μg/ml (500ng/ml). Immediately add 100μl per well. Incubate at room temperature for 2 hours.

Avidin-HRP Conjugate: Aspirate and wash plate 4 times. Dilute Avidin-HRP conjugate 1:2000 in diluent.
Add 100μl per well. Incubate 30 min at room temperature.

ABTS Liquid Substrate: Aspirate and wash plate 4 times. Add 100μl of substrate solution to each well.
Incubate at room temperature for color development. Monitor color development with an ELISA plate reader at 405 nm with wavelength correction set at 650 nm.

NOTE: Reliable standard curves are obtained when O.D. readings do not exceed 0.2 units for the zero standard concentrations, or 1.2 units for the highest standard concentration. The plate should be monitored at 5 minute intervals until desired O.D. readings are obtained. The typical range is 5-40 minutes. O.D. readings may vary.

NOTE: antibody ab9570 can be paired for Sandwich ELISA with https://www.abcam.com/index.html?datasheet=83132. (To detect hVEGF by sandwich ELISA (using 100 µl/well antibody solution) a concentration of 0.5 - 2.0 µg/ml of this antibody as Detection is required. This antigen affinity purified antibody, paired with the above recommended pair, allows the detection of at least 0.2 - 0.4 ng/well of recombinant hVEGF.)

------------------------------------------------------------------------

For neutralization we have the following information:

To yield one-half maximal inhibition [ND50] of the biological activity of hVEGF (10.0 ng/ml), a concentration of 0.05 - 0.1 µg/ml of this antibody is required.


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

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Answer

Thank you for contacting us. Unless we specify it on our datasheet, all the antibodies work under denatured, reducing conditions. However, this antibody should work under both reduced and non-reduced conditions. I would suggest repeating the assay using denatured samples prepared following the WB protocol form our Website. This protocol is very interesting as it comprises all the required steps to perform a WB assay, including sample preparation. https://www.abcam.com/index.html?pageconfig=resource&rid=11375 If the results don’t improve after following the suggested protocol, please contact me again and I will be happy to help you further.

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1-10 of 13 Abreviews or Q&A

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