Anti-nNOS (neuronal) (phospho S847) 抗体 (ab16650)

Thank you for the extra details of your protocol. I think there is nothing wrong with your protocol. I was concerned that you may have been using milk to block the membrane and dilute the antibody, since this can sometimes interfere with detection of phospho-proteins. The Odyssey blocking buffer does not, so I think we should consider replacing this antibody.

Can you please send me the order number (either a PO number or the Abcam reference number), or the name of the person who placed the order, the institution, and the approximate date?

Thank you for bringing this to our attention.

Can you please confirm that you are using ab16650?

I think there are too many bands to identify which is nNOS, but if you send a few details of your western blot protocol, I may be able to suggest a modification that improves the result.

Can you please tell me:

1. How much sample you load per lane of the gel?
2. What you are using to block the membrane?
3. What you dilute the antibody in?
4. What concentration or dilution of antibody you incubate with the membrane?

I look forward to your reply.

Thank you for taking time to complete our questionnaire. I am sorry to hear that this antibody is not providing satisfactory results.

The details provided will enable us to investigate this case and will provide us with vital information for monitoring product quality.

Having reviewed this case, I would like to offer some suggestions to help optimise the results from ab16650 Anti-nNOS (neuronal) (phospho S847) antibody. I would also appreciate if you can confirm some further details:


1.As you are fixing with PFA and overnight, a gentle antigen retrieval may be necessary. Please reffer to the following http://www.elsevier.com/wps/find/journaldescription.cws_home/506079/description#description. 2000 Dec 15;104(1):27-34.

2. What kind of incubation buffer are you using? Does it contain a detergent like Tween or Triton? If you don't Iwould like to suggest toadd 0.025% Triton X-100 to facilitate the antibody solution.

3. I would like to suggest to block the endogenous biotin as you are working with the ABC system. this may explain the high background of the old lot.

4.Have you alternated the antibodies concentration? According to the datasheet, the antibodies concentration fluctuates form lot to lot.


Should the suggestions not improve the results, please do let me know.

In the event that a product is not functioning in the species and applications cited on the product datasheet (and the problem has been reported within 6 months of purchase), we would be pleased to provide a free of charge replacement, credit note, or refund.

I hope this information is helpful, and I thank you for your cooperation.

I am very pleased to hear you would like to accept our offer and test ab16650 and ab5583 in electric fish. This code will give you 1 free primary antibody before the expiration date. To redeem this offer, please submit an Abreview for electric fish and include this code in the “Additional Comments” section so we know the Abreview is for this promotion. For more information on how to submit an Abreview, please visit the site: www.abcam.com/Abreviews. Remember, we publish both positive and negative Abreviews on our datasheets so please submit the results of your tests. The code will be active once the Abreview has been submitted and can be redeemed in one of the following ways: 1) Call to place your order and mention the code to our customer service department; 2) Include the code in your fax order; 3) Place your order on the web and enter the promotional code. Any feedback that you can provide will be greatly appreciated, whether positive or negative. If you have any further questions, please do not hesitate to contact us. We look forward to receiving your Abreview and wish you luck with your research. The terms and conditions applicable to this offer can be found here: www.abcam.com/collaborationdiscount.

Thank you for the protocol details that you have provided. I would expect you to see a band at approximately 160 kDa using mouse forebrain samples and using ab16650 at a concentration of 1 ug/ml. Since you are not seeing anything at all, even with the primary at 2 ug/ml, incubation o/n at 4C and up to 80 ug of lysate loaded, it definitely seems as if there is a problem with the vial that you received. I just want to check, do you know that your secondary is working properly with other primaries? I would be happy to offer you a free of charge replacement vial or can offer to issue you a credit note or refund. Please let me know how you would like to proceed and I look forward to hearing from you.