Anti-HIV1 tat antibody [1] (ab42359)

Dear Abcam,   We are having unspecific staining with your tat antiboy.   We tested arrays of HIV negative tissues and we got membrane and cytoplamic staining not expected at all.   So the initial result  on lymphones taken from not  treated HIV patient was promising but when we tested it on negative control tissues as tonsils guts lymphonodes and liver we got the same staining result!   I look forward to hearing from you.   Best regards,  

ANSWER:

Thank you for contacting Abcam.

I am sorry to hear of this issue with our product, especially after your earlier work seemed to have exciting implications. This product is very well mapped and I am surprised to hear of the results that you have seen you have tested in multiple negative (uninfected) control tissues in IHC-P.

Would you be able to send me detailed information about your protocol? It would also be very helpful to see images of your stains. With this information I may be able to offer suggestions that may help or to offer a replacement or refund if we cannot resolve these issues.

I look forward to your reply. Please let me know if you have any other questions.

Hi  

if you have a look at this link:

  http://www.uniprot.org/uniprot/P04610  

you scroll down up to Subcellular location   Host nucleus › host nucleolus. Host cytoplasm. Secreted. Note: Probably localizes to both nuclear and nucleolar compartments. Nuclear localization is mediated through the interaction of the nuclear localization signal with importin KPNB1. Secretion occurs through a Golgi-independent pathway. Tat is released from infected cells to the extracellular space where it remains associated to the cell membrane, or is secreted into the cerebrospinal fluid and sera. Extracellular Tat can be endocytosed by surrounding uninfected cells via binding to several receptors depending on the cell type.  

given that our lymphonodes test sample is from an HIV patient (not treated as well) that would explain why we see strong membrane staining.  

What about?  

Thank you.  

ANSWER:

Thank you for your reply.

The information that you have sent and the tissue that you are staining on would seem to explain the membrane staining nicely. I certainly do appreciate your finding this for me and will add this information to our notes.  If you perform any staining on test samples of HIV infected but treated patients would you please let me know the results?

Thanks again. If there is anything else that we can do for you, please do not hesitate to contact us.

I just wanted to know if  is also localized in the cytoplasm and not just in the cell nuclei.   Do you have any evidence for that?   In the literature I did find reports of Tat being expressed also in the cytoplasm. So I am investigating in that direction now.   My test sample is paraffin embedded tissue (lymphonoides from an infected and not treated HIV patient).    The staining if specific is fine then. Plenty of brown positive cells.   Thank you.  

ANSWER:

Thank you for your responce. I have been unable to find data showing HIV1 tat in the cytoplasm. I am sorry that I could not be of more help.  I would recommend using positive and negative controls to ensure that your results are accurate. I would like to direct you to the HPA link which has some excellent images.

http://www.proteinatlas.org/ENSG00000102241

Please let me know if you have any questions or if there is anything else Abcam can do to help you meet your research goals.

Dear Abcam   We bought recently the anti tat antiboby  (ab42359) and I have a question for you:   we saw very good immunostaining on our test sample (HIV infected lymphonodes) but it was a cytoplasmic/membrane antigen stain   Your Ab data sheet says Cellular localization Nuclear. Upon stimulation with EDN1, it is exported from the nucleus to the perinuclear region.   Any idea about? Is the staining specific?  

ANSWER:

Thank you for contacting us. I am sorry to hear you are experiencing difficulties with one of our products. We take product complaints very seriously, and investigate every product that we feel may not be performing correctly. I would like to gather further information regarding the samples tested and the protocol used to see if there are any suggestions we can make that may improve the results. This information will also allow us to investigate this case internally and initiate additional testing where necessary. If the product was purchased in the last six months and is being used according to our Abpromise, we would be happy to replace or refund the antibody. Could you please send me your protocol, including but not limited to:   Tissue preparation and fixation procedures. Any permeablization or antigen retrieval steps that you use. Blocking solution compositon, blocking time and temperature. Concentrations of the antibodies that you have tried, how long they were incubated and at what temperature. Any positive control tissues that you have used and what the results where of those. Any negative controls that you performed. Any images that you may have will be of great help as well. Thank you in advance for collecting this information for me.  If you have any questions please feel free to contact me. I look forward to receiving your reply.