Anti-VEGFC antibody (ab9546)

The samples were reduced SDS extracts of trachea from VEGF-C over-expressing mice. The ˜ 55- 65kDa band(s) is present in the over-expressing mouse and not the control litter-mate. This would fit the predicted size of unprocessed mouse VEGF-C with glycosylation heterogeneity.

ANSWER:

Following up my previous e-mail, I received the following from our source for this antibody:

"The proteolytic processing of mammalian VEGF-C is a complicated step and described before. Therefore the work by Joukov et al. (EMBO J 16, pp3898-11, 1997) must be read very carefully. The antibody made against the mature VEGF-C region will certainly recognize the full unprocessed, partially processed and mature VEGF-C. If you take whole mouse tissue you may come up with the detection of the more intracellular unprocessed or partially processed forms and not so much the released forms fully processed by (extracelluar?) proteases. Cell culture supernatants from mouse trachea cells will probably give a total different picture."

Please let me know if you have questions or concerns.

Western blot band appears at 55 kDa, not at the expected 20 kDa,concerned that the antibody stains immature protein preferentially.

ANSWER:

Thank you for bringing this to our attention. I am waiting for the lab to send details of the immunogen for the VEGF-C antibody.

I am skeptical that the immunogen contains the propeptide sequence, but in any case, I want to help you make sense of your results. Can you please describe your samples: tissue or cell type, and how they are prepared for SDS-PAGE? Do you have any reason to believe the 55 kDa band might be VEGF-C, for instance differences among band intensities that correlates with treatment?

Hi,

I've bought several antibodies from abcam (ab2074, ab9546, ab9797..) to detect the expression of these proteins in animal tissue by IHC-P.

Follow the protocol on  abcom webside,  recommended that," blocking of endogenous peroxidase can be performed after the primary antibody incubation". and the reason is "Some epitopes are modified by peroxide, leading to reduced antibody:antigen binding. Incubating sections with peroxide after the primary incubation avoids this problem."

My question is, since the peroxide can modify some epitopes, can it also modify the primary antibody, and then reduce the binding of primary antibody and second antibody? did you have made some experiment to confirm it? By the way ,can the hydrogen peroxide be diluted in PBS instead of TBS?

ANSWER:

Thank you for contacting us.

While it is true that the H2O2 solution may have a small effect upon the secondary antibody recognition, this effect cause limited or no issues with your staining as the structure of the primary antibody allows for multiple recognition sites for the secondary.

In regards to using TBS in place of PBS, I have found no mention of it as an alternative to PBS for this solution and as such cannot recommend it.


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

For the Vegf-C there appears to be a large range that the anti-vegf-c antibody covers (from about 15kDa to 24kDa). It is stated in the information on Vegf-c (isolated in rabbit) that it is supposed to be 23kDa, were the protein samples in the gel that was used for this western spread out? Why is the range so broad? Thank you for your time Brian

ANSWER:

We used both recombinant rat and human dNdC-VEGF-C. The material is partially unglycosylated as well as glycosylated to different degrees. We do not have a Western blot image for Hif-1 alpha.

We ordered the following antibody from abcam: #ab9546 anti-VEGF C We are using the antibody for western blots. In the product sheet sent with the antibody, you recommend using it at a 1mg/ml concentration. Is this right? That is a very high concentration. And, you only send .25mg of the antibody total in the sample to begin with. Please verifiy that this is the concentration that is to be used for Western Blots.

ANSWER:

The concentration is incorrect, it should be 1ug/ml and not 1mg/ml. I have corrected the datasheet and competely updated it (as it was one of our older ones) so now it should be much clearer. Please check the new on line datasheet. Thank you for bringing this mistake to our attention and we apologise for any inconvenience caused.