Anti-VEGF antibody (ab9570)

Hello, I am using ab9570 anti-rhVEGF antibody to detect 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 the manufacturer 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?

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.

I am interested in VEGF165 (recombinant expressed in E. coli) as well as in an Anti-VEGF-antibody. I would like to do conformation analysis and I need the mature VEGF-protein with length of 165 amino acids. So there should be no N-terminal signal sequence.

You offer these three VEGF165 proteins: ab56620, ab61861, ab9571 and I wonder which of your products is the right one for me. Do ab56620 and ab61861 contain 165 or 191 aa?

Further which antibody (preferred host: rabbit) do you recommend to detect the mentioned proteins?

Can you give me any further sequence information about the target proteins of following antibodies: ab52917, ab9570, ab99511?

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.

crossreactivity with other growth factors tested for both Abs?

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.

Please send protocols for ELISA, sandwich ELISA, neutralizaion, and western blotting.

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.
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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.
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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 http://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.)

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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.

Hello, Our labrecently bought ab9570, an antibody to VEGF, for use in Western Blotting. I didn't see on the data sheet if it was intended for use with reduced or non-reduced VEGF samples, so I figured it was for reduced samples. I didn't get any bands, so we did a dot blot with native, non-reduced and reduced VEGF, just to see what it would recognize. It did not detect the reduced, but did detect the non-reduced and even more strongly detected the native VEGF. Was the figure in the data sheet for the WB used under non-reduced condition? Samples are never run in native form right?

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. http://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.