Anti-PARK7/DJ1 antibody [malphaDJ-1/E2.19] (ab11251)

Previous vial of ab11251 worked in IHC-P, lot 63399 (liquid in vial clear). Vials states Ascites
New vial of same antibody lot 63399 is not working in IHC-P, liquid in vial is yellowy.Vial does not state ascites.

ANSWER:

Thank you for contacting us and reporting the problems you have encountered with ab11251. Unfortunately xxx is away today but she has asked me to get in contact with you.

xxxxhas been investigating how the antibody is produced and purified in house and why there may be a difference in the two vials that you have received and shewill get back to you with this informationas soon as she hasit. In the meantime, if you wouldn'tmind, could you provide a few moredetails of whatproblems you havebeen experiencing with this antibody?I have attached aquestionnaire to this email for this purpose. Thiswill help greatly in investigating this case further and initiating any additional in house testing where necessary. Ifyou could provide images of theantibodyworking andof the latest results that wouldbevery useful.

As discussed with Kate, although this antibody has not been tested by us for paraffin embedded IHC and would therefore not normally be covered by the Abrpomise, as youhave previously found it to besuccessful in this application we can extend the Abpromise guarantee as a good-will gesture. I am thereforeable to offer you areplacement of thisantibody with adifferent lot,a different antibody altogether (such as http://www.abcam.com/PARK7-DJ1-antibody-ab18257.htmlwhich has been used in paraffin embedded IHC) or if you would prefer, a refund.

I apologise for the inconvenience this problem has caused and look forward to hearinghow you wouldlike to proceed.

1 In which dilution the antibody will works best 2 Does the ab11251 fit for immunoprecipitation? thanks for your reply.

ANSWER:

Thank you for your enquiry. Ab11251 has been tested for application in Western blotting and Immunocytochemistry, and has not yet been tested in any other applications (including IP). For Western blotting, I would recommend starting at a dilution of 1:500, and for ICC I would recommend starting at a dilution of 1:100. Please optimize based upon your results.

If you decide to go ahead and purchase this product, please let us know how you get on by submitting an Abreview and in return we will award you 50 Abcam Points, which can be redeemed on a number of rewards (a further 100 Abcam Points will be offered for an image).

If you have any additional questions, please contact us again.

BATCH NUMBER -- NOT SPECIFIED -- ORDER NUMBER 66263

DESCRIPTION OF THE PROBLEM No staining in the brain tissue, staining of the skin

SAMPLE zebrafihs whole mount 1-2 days post fertilisation

PRIMARY ANTIBODY DJ-1 ab11251

SECONDARY ANTIBODY ABC kit Vector Labs (the dilutions are tested using other antibodies, works nicely)

DETECTION METHOD DAB

POSITIVE AND NEGATIVE CONTROLS USED omission of the primary and secondary antibodies

ANTIBODY STORAGE CONDITIONS reconstituted in water, stored in aliquotes at -20. once thowed, stored at +4.

FIXATION OF SAMPLE 4% PFA

ANTIGEN RETRIEVAL none

PERMEABILIZATION STEP methanol, followed by incubation with PBS+0.3% Triton X100+1%DMSO

BLOCKING CONDITIONS same as permeabilization + 4% NGS

HOW MANY TIMES HAVE YOU TRIED THE APPLICATION? 3-4 HAVE YOU RUN A "NO PRIMARY" CONTROL? Yes DO YOU OBTAIN THE SAME RESULTS EVERY TIME? Yes

ADDITIONAL NOTES you indicate that this antibody crossreacts with zebrafihs tissues. Could you please specify the conditions and methods used.

ANSWER:

Thank you very much for your enquiry and for your patience. Ab11251 was originated outside Abcam and according to the originator, they had positive feedback from a researcher who found the antibody to cross-react with zebrafish. That is unfortunately all the information that I have regarding the antibody's cross-reactivity with zebrafish.

I saw that you are using ab11251 with PFA fixed brain tissue. To our knowledge, this antibody has only been tested for application in Western blotting and Immunocytochemistry. We don't have any information about how ab11251 works in IHC.

At this point I would like to make the following suggestions. The PFA fixative may be modifying the epitope that the antibody recognizes and so I would suggest doing an antigen retrieval step in order to unmask the epitope. Also, you didn't mention which dilutions of ab11251 that you have tried but you may want to increase the concentration.

If you have any additional questions, please contact us again.