Anti-Rab25 antibody (ab45855)

Hi,   I have used the antibody at twice that concentration and not been able to detect Rab25. I am suspicious as I cannot detect over-expressed Rab25 using that antibody. I also know that the over-expressed version on Rab25 in A549 cells runs at the correct size (25kDa). How have you verified that the band at 32kDa is really Rab25 that is post translational modified?   I do agree that A549 cells probably express very low levels of Rab25 and that could be the problem. However, I do get a very interested “Rab25” stain using only that antibody (not with any of your other Rab25 antibodies) and thus must be one of the unspecific proteins. I would really need to know what that antibody recognizes.  

ANSWER:

Thank you for your reply. I have talked to the lab to see if they have any more information about the unspecific proteins that ab45855 recognizes. Unfortunately they have not been able to determine the identification of these bands. They did inform me that for this antibody, it may be more effective to use BSA rather than milk as a blocking and dilution agent, this could help to detect the band of interest. As ab45855 is guaranteed to work in human samples and in western blot, if using BSA does not prove to be successful then I would be happy to find an alternative Rab25 antibody that would be suitable for you and send it to you free of charge.

Please let me know how you would like to proceed and if there is anything else I can do to help do not hesitate to contact me.

Hi, I have a problem with this antibody and I need your input on it. I have tried to detect Rab25 using it, even after over-expression of Rab25, and not been able to detect any proteins at the right size. The only proteins that I do recognize are the ones at 17 and 46 kDa on a Western blot. I do know that Rab25 is running perfectly at 25kDa using other antibodies so the suggestion that Rab25 is posttranslational modified and running at 36kDa is probably not the case in my cell line (A549). Do you have any idea about the additional proteins and what they might be? I am suspicious about the quality of that antibody, does it recognize the peptide used to immunize the animals? The last thing that I did was to use one entire of vial of antibody (100μg) in a pull-down experiment to try to figure out what proteins the antibody recognizes. I was not able to detect any proteins except the antibody itself using an Imperial protein stain. I am stuck and would appreciate any help.   All the best,

ANSWER:

I am sorry that you are experiencing issues with this antibody. This particular antibody does recognize unspecified bands at 16kDa and 45kDa, and we are still trying to identify what those bands are, but it should also recognize a band at 32kDa, which is Rab25, that has undergone post-translational modification. I know you say that you do not see this band is not seen in your samples and I was wondering if you had run other samples along with your A549 sample, maybe MCF7, HeLa cells or even tissue samples, liver, testis stomach all have high levels of Rab25. I am just curious if this antibody is just not recognizing the correct band in your samples or in any samples. Also, what concentration were you using the antibody at? From the data on the datasheet, it seems as though you have to use 1ug/ml of antibody, with 10ug of total protein loaded, even then the 32kDa band is fainter that the others and so a longer exposure would be necessary. As for the IP results, the antibody had not been tested in IP previously and so it may not be suitable for that application, which may be why there were no proteins associated with it.

We do guarantee all of our antibodies for 6 months (when stored according to the data sheet) and this antibody is guaranteed to work in western blot on human samples. Therefore if we cannot resolve the issue, then I would be happy to see if another antibody will fit your needs, or process a refund.

I look forward to your reply and if there is anything else I can help you with, please let me know