Anti-Dnmt3b抗体(ab2851)

Thank you for confirming these details and for your cooperation. The details provided enable us to closely monitor the quality of our products.

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 replacement with ab2851. This will actually ship today and you should receive it tomorrow.

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 for contacting us and taking the time to send the questionnaire. We really appreciate all the details provided as well as the images sent, that help us to better understand the problem. All of our products are covered by our Abpromise guarantee; which ensures that you can trust our products, and they should work in the tested species and applications stated on the datasheet, or we will offer a replacement, credit, or refund, if reported within 6 months of purchase. I would really like to help you solve the problem and obtain good results from this antibody, and for that I am very pleased to give you some protocol tips. In the case these suggestions don’t improve the results, please let me know, and I will be happy to send a replacement or a credit note. -In order to enrich the signal, prepare nuclear lysates to increase the protein levels in the sample. -During sample preparation, it is crucial to use protease inhibitors as well as phosphatase inhibitors to avoid sample degradation. -I would suggest trying a different blocking agent. Have you tried 5%BSA for 1-2 hours at RT? -Decreasing the secondary concentration may also help to improve the signal. -To avoid smearing of the gel, using fresh prepared buffers is crucial. - You may run a positive control to assess how well the antibody is working. This protein is highly expressed in JEG-3 lysates. I hope these tips help, thank you for your cooperation. Should the suggestions not improve the results, please do not hesitate to contact me again, and I’ll be more than happy to help you further.

A brief literature search revealed that PMID: 16357870 (Vire E et al Nature 2006 439:871) has used U2OS cells and primers specific for the hsMYT1 promoter. The primers are described in PMID: 15231737 (Kirmizis A et al Genes and Dev 2004 18:1592). I hope this information will be useful,

Thank you for getting back in touch with me. To clarify. A predicted molecular weight of 97KDa for Dnmt3b is derived from SwissProt reference Q9UBC3. This is purely based on the protein sequence and a prediction. ab2851 detects a band of approximately 130 kDa which correlates with the Dnmt3b reference that you have provided me with. I can tell you that this antibody also detects recombinant DNNMT3b1 at ~130KDa and recombinant DNMT3b3 at ~100 kDa; this may explain the additional bands. I hope this information helps, please do not hesitate to contact us if you need any more advice or information.

I have been advised by the source of ab2851 that it would be most important to run a positive control. In the lab recombinant DNMT3b has been used, however it is reported that DNMT3b is highly expressed in P19 (murine EC) cells when compared with human cells such as HeLa or HCT116. I would therefore recommend to run P19 cell lysate. It would be worth trying to block the membrane to see if the BSA alters the banding pattern. It would also be worth running a negative control (no primary antibody) to determine if the non-specific banding is being caused by interaction with the secondary antibody. An interesting point was raised by my colleagues: splice variants: It is known that there are at least 5 different splice variants for DNMT3b, and it is also believed that the protein is post-translationally modified. Therefore it is not unexpected to see some size heterogeneity. Finally, I may be able to add to the catalogue some neutralizing peptide to determine which bands are specific if you are interested in trying this. I hope the advice detailed above is useful, please do not hesitate to contact me if you have further questions,