Anti-Nav1.6/SCN8A抗体(ab65166)

Thank you very much for your reply, and I am very sorry for all of the trouble with these antibodies.

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Please let me know if you have any questions or if there is anything else that we can do for you.

Thank you for sending the additional information and for your patience.

I forwarded your protocol details to the lab for a second look, and we would have expected the antibody to work in the conditions that you've described. I am very sorry that neither of these antibodies has worked for you, and I can arrange a credit or a refund for your original purchase if you'd like. Alternatively, we do have one other antibody that is predicted to work with rat samples, ab135457-

https://www.abcam.com/Nav16-antibody-ab135457.html

This antibody hasn't been tested with rat samples, but since the immunogen is homologous to the rat protein it may work. I'd be happy to send this out if you'd like to try it.

I apologize again for the poor results with these Nav1.6 antibodies, but if there is anything else that we can do for you, please let me know.

Thank you very much for keeping me updated about the results with ab65166.

I'm sorry to hear that there was still no band on the blot. Would you mind telling me some more about the protocol you used with ab65166?

1) Are you still using rat hippocampus lysates? How much protein was loaded per lane of the gel?

2) Could you describe the electrophoresis and gel conditions (% of the gel etc.)?

3) Could you describe the transfer and blocking conditions (buffer, transfer time, blocking agent etc.)?

4) Does the secondary antibody work with other primaries?

5) Detection method (ECL, ECLPlus etc.)

6) Did the ladder proteins transfer, or did you confirm transfer of the high molecular proteins via Ponceau stain?

We don't have any other antibodies to Nav1.6 that have been tested in Western blot with rat lysates, but I'll see if there are any protocol suggestions that we can make. If the results are still not satisfactory, we can arrange a credit or refund for your original purchase.

Please let me know if you have further questions or comments, and I'll be happy to help.

Thank you very much for your calls today and for letting us know about the trouble with ab83764 in Western blot.

Please keep me updated about the results after using ab65166 with your samples.

I look forward to hearing from you. If you have any questions or if there is anything else that we can do for you, please let me know and I'll be happy to help. Have a nice weekend!

I am sorry to hear that you have been experiencing problems using this product in the application that you wish.

In order to assess the quality of our products I would ask that you complete a brief questionnaire relating to the application used. Often it is possible to make suggestions that may help resolve problems experienced using a particular product.

As our Abpromise indicates, in the event that a product is not functioning in the applications/species cited on the product data sheet (and the problem has been reported within 6 months of purchase) we will happily offer a credit note/refund to the value of the product purchased.

All our customer feedback, including complaints are monitored weekly by our in house technical support team. If a product is at fault the technical support team will consider removing the product from our catalogue in order to avoid future customer inconvenience.

Could you please provide some further details of the protocol used and complete the following form (attached as a word document). It would be much appreciated if you could attach an image to the response.

Thank you for your understanding and co-operation in this matter. I look forward to hearing from you soon and resolving this issue as soon as possible.

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 the order number xxx. The estimated delivery date is xxx as the item is currently backordered. I hope this is not causing a big problem. I apologize.

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 your reply.


I have a few additional suggestions. I am not sure if my colleague had mentioned those to you:

1) You can check the transfer with Ponceau S (which is a reversible membrane stain) to be sure that you transfered the proteins. This will give you also an additonal quality control for the transfer. My feeling is that transferingat 75V for 4 h could be too long. if the gel can withstand a higher voltage, I'd suggest to try 100V for an 1 h at 4 degree with stirring.

2) Further, I'd suggest to use 10% MeOH and reduce the amount of SDS to 0.1% in the transfer buffer. You want to have some SDS to prevent hydrophobic proteins (like Nav1.6) from precipitating in the gel during the transfer, but 0.5% seems a bit high.

3) Are you blotting for any other proteins, such as actin or tubulin as loading controls? This would be another way to check if the WB protocol in general works and if the mouse brain lysate you have is OK.

4) How are your samples lysed? I would recommend using RIPA buffer since the Nav1.6 protein is very hydrophobic.

I also checked online and found the following link for WB with membrane proteins:
http://www.sciencedirect.com/science/article/pii/S0003269708006829

Our WB protocol guide can be found here: https://www.abcam.com/index.html?pageconfig=resource&rid=11375and I have it also attached to this email.

To answer your question about the lysate:

I'd be happy to send you another vial of ab30151 free of charge. Alternatively, we also have ab7188 (Brain (Mouse) Tissue Lysate - normal tissue, 0 days old) [https://www.abcam.com/Brain-Mouse-Tissue-Lysate-normal-tissue-0-days-old-ab7188.html] in the catalog, but I am not certain if newborn mice express this protein strongly.
I can send you 1 vial of either one or the other free of charge. Please let me know which one you would prefer. Please let me know your order or PO number for the lysate.

I look forward to hear back from you and assist you further.

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Thank you for calling and bringing this issue to our attention.

I just wanted to make sure you were loading the correct amount of protein and to make another suggestion regarding the blocking step.

For the brain lysate, ab30151, you will want to load at least 10ug, which is a volume of about 4 microliters.

For the blocking step, you may find that a relatively short block (30 minutes) instead of overnight, followed by overnight incubation with the antibody will help develop the signal, compared to an overnight block followed by a shorter incubation with the primary. These are little modifications, though. I suspect that either the lysate is defective or the transfer was not optimal. Please let me know how your tests go. If they fail, we will be happy to discuss a replacement of either the antibody or the lysate, or both.