Anti-Bmi1抗体(ab38295)

Thank you for your response.
I have discussed this enquiry with my colleagues.
A BLAST with the immunogen in the human proteome does not predict cross-reactivity with any other proteins, just BMI-1 and partial BMI-1 sequences. It could be a cleavage product, but I have been unsuccessful in finding a publication that shows this.
According to Swiss-Prot database, the intracellular localisation of Bmi1 can be both nuclear and cytoplasmic. You may find some further information at this site:
http://www.uniprot.org/uniprot/P35226
1) "E4F1: a novel candidate factor for mediating BMI1 function in primitive hematopoietic cells."
Chagraoui J., Niessen S.L., Lessard J., Girard S., Coulombe P., Sauvageau M., Meloche S., Sauvageau G.
Genes Dev. 20:2110-2120(2006) [PubMed: 16882984] [Abstract]
Cited for: FUNCTION, INTERACTION WITH E4F1, SUBCELLULAR LOCATION.
2) "Interaction proteomics analysis of polycomb proteins defines distinct PRC1 Complexes in mammalian cells."
Vandamme J., Volkel P., Rosnoblet C., Le Faou P., Angrand P.O.
Mol. Cell. Proteomics 0:0-0(2011) [PubMed: 21282530] [Abstract]
Cited for: IDENTIFICATION IN A PRC1-LIKE COMPLEX, SUBCELLULAR LOCATION.
Please see attached the image of Bmi1 staining with FFPE rat thymus sections.
If you need any further assistance in the future, please do not hesitate to contact me.

Thank you for your enquiry regarding ab38295 and for taking the time to provide some useful details of the experiments. I am very sorry to hear that you are having problems with this antibody.
My colleague has asked me to look after her customers while she is busy with other projects. I apologize for my delayed response.
Though you have kindly provided some details, it would be much appreciated if I could get some more information which would help me identify the source of the problem.
1) Fixation: stomach tissues were fixed in 4% formalin for 2
hours, followed by 1 hour’s wash in tap running water before being
dehydrated.
- Question: Would you be so kind to confirm the purpose of washing the fixed tissue in tap water? Normally, for rehydration it is recommended to use TBS-T.
https://www.abcam.com/index.html?pageconfig=resource&rid=11384
https://www.abcam.com/index.html?pageconfig=resource&rid=13046
2) Antigen retrieval:
retrieval was performed in boiling citrate
buffer (ph6) for 15min
- Question: Has the time for AR been optimized and so what equipment was applied (microwave, steamer, vegetable cooker etc)?
Three minutes is only suggested as a starting point antigen retrieval time. Less than 3 minutes may leave the antigens under-retrieved, leading to weak staining. More than 3 minutes may leave them overretrieved, leading to non-specific background staining and also increasing the chances of sections dissociating from the slides. A control experiment is recommended beforehand, where slides of the same tissue section are retrieved for 1, 2, 3, 4 and 5 minutes before being immunohistochemically stained to evaluate optimum antigen retrieval time for the particular antibody being used.
https://www.abcam.com/index.html?pageconfig=resource&rid=11488#2
Thank you for your understanding and co-operation in this matter. I look forward to hearing from you and hope to solve this problem as soon as possible.

Vielen Dank für Ihre Anfrage.
Es tut mir Leid zu hören, dass Sie Probleme mit diesem Antikörper haben. Laut unseren Unterlagen haben wir den Antikörper nicht selber in der IHC getestet, sondern haben diese Applikation aufgrund einer Publikation aufgenommen.
Ich habe unseren Fragebogen als Word-Dokument an diese E-Mail angehängt. Durch das Ausfüllen des Fragebogens erhalten wir alle nötigen Informationen über Ihre Proben und Ihr Protokoll. Sobald Sie dieses Formular an uns zurückgeschickt haben, werden wir uns Ihr Protokoll ansehen und möglichst Veränderungsvorschläge machen, die Ihre Ergebnisse verbessern werden.
Falls sich allerdings bestätigt, dass der Antikörper nicht so funktioniert, wie auf dem Datenblatt beschrieben und er innerhalb der letzten 180 Tage gekauft wurde, werden wir Ihnen gerne einen Ersatz oder eine Gutschrift schicken.
Ich freue mich, bald wieder von Ihnen zu hören.

I am sorry this product did not perform as stated on the datasheet and for the inconvenience this has caused. As requested, I have issued a free of charge replacementcontaining 1 vialeach of ab38237,13533,38295.

To check the status of the order please contact our Customer Service team and reference this number.

Please note that this free of charge replacement vial is also covered by our Abpromise guarantee. Should you still be experiencing difficulties, or if you have any further questions, please do not hesitate to let us know.

I wish you the best of luck with your research.

Thank you very much for following this up with the customer. I am sorry that the change in the blocking buffer did not improve the results.

I would still like to recommend to the customer to reduce the samples for the Western blot either with DTT or beta Mercaptoethanol. Indeed, not all proteins need to be reduced to be visible in the Western blot and I am not sure whether it is necessary to reduce the samples to see a nice band of BMI1. As however the BMI1 is a highly complexed protein (http://www.uniprot.org/uniprot/P35226) and we have tested it under reduced conditions, I think there is the possibility that reducing the samples could solve the problem.

As I understand howeverthe frustration the customer must be experiencing, I will provide a credit note for this antibody. Please do let me know whether the customer would like to accept this.

Thank you for your cooperation. I am looking forward to hear back from you.