Anti-Notch 2 antibody (ab8926)

problem with ab14955 in IHC-P, Mouse samples.

Would like to test ab8926 on Mouse tissue (untested species)

ANSWER:

Thank you for calling us and for alerting us to the problem you are experiencing with our product. We take product complaints very seriously, and investigate every product that we feel may not be performing correctly.

As we discussed on the phone, I am attaching our questionnaire so that we can gather further information regarding the samples tested and the protocol used. Once we receive the completed questionnaire, we will look at the protocol and 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.

About the Notch 2 antibody ab8926 : To our knowledge, ab8926 has not been tested in mouse. Therefore, I can offer a discount off a future purchase if you buy ab8926 now, test it in Mouse and submit feedback to us in the form of an Abreview. It doesn’t matter whether the Abreview is positive or negative, we would just really like to receive your feedback. The discount would be to the value of 1 free primary antibody.

If you are interested in this offer, please follow these steps:

1. Reply to this e-mail to let me know that you would like to proceed and test ab8926 in mouse. I will then send a discount code. This code must be issued before purchasing ab8926 so please wait for my reply before ordering.

2. Purchase ab8926 either by phone, fax, or online (www.abcam.com).

3. Test it in mouse.

4. Let us know the results, positive or negative, using our Abreview system (this will take about 10 minutes and images are great if you have them!). To find out how to submit an Abreview, please visit: http://www.abcam.com/abreviews.

5. After the review is submitted to us, the discount code becomes active. Simply place your new order by phone, fax, or on the web and mention the discount code. The discount can be redeemed for any primary antibody ordered and the discount code is valid for 4 months after issue.

We are always pleased to obtain feedback about our products and any information is greatly appreciated! Even if ab8926 turns out to be unsuitable for mouse, you will still receive the discount on your next purchase after your Abreview has been submitted.

Please let me know if you have any questions about this offer and I would be happy to help you further.

The Terms and Conditions of this offer can be found at: www.abcam.com/collaborationdiscount.

I look forward to receiving your reply with your questionnaire and image(s) for ab14955.

I submitted Abreview about ab8926 with a WB in TIFF file last week. Please check. You may see how GSI affect Notch2 cleavage status. I am actually confused. Let me know what you think ASAP. Thanks!

ANSWER:

When I was a postdoc I studied gamma-secretase inhibitors, APP, abeta, Notch etc. so I feel like I can interpret the result. I would have expected the activated Notch2 protein to be ~ 77 kD more or less. 20 uM of most gamma-secretase inhibitors if applied the correct way should inhibit Notch cleavage. What gamma-secretase inhibitor are you using and how did you apply it? What cells are you using? Have you tried blocking in 5% milk for an hour and then incubating in primary overnight instead of only for 2 hours? I would predict a band at about 77 kD and then as you apply more of the GSI the band should disappear. If the antibody also recognized the uncleaved Notch2 then uncleaved Notch2 should accumulate, but this antibody should only recognize activated Notch2.

Strangely the review seems to demonstrate that the 73 kD band accumulates and the 23 kD band disappears. Based on MW and this being an activated Notch2 antibody I would think both bands are non-specific, but it is very strange that these two non-specific bands respond to GSI treatment.

What is your interpretation?

I look forward to your reply.

Hello, Bill:

When I used gradient gel for WB, your ab8926 gave me total 3 binds, 2 looks like dimer around 60kDa, the other smaller one around 23kDa, gamma-secretase inhibitor blocked the 2 bigger binds cleavage. But I am not sure the antigen/immunogen you used was only the most active NICD or may be a larger fragment which including part ot transmembrane. Would you tell me the size of protein you would predict using your ab8926? Thanks!

ANSWER:

Thank you for your reply.

I was a postdoc in Weiming Xia's lab for four years and spent a considerable amount of time conducting research on gamma-secretase so I should be able to help you. The immunogen we used was CRDASNHKRREPVGQD, corresponding to amino acids 1719-1733 of Human activated Notch2. The Notch 2 intracellular domain is from amino acids 1697 to 2471 and is 775 amino acids long. The transmembrane domain is potentially from amino acids 1678 to 1698. Im would have expected the NICD2 band to be ~ 77 kDa, however, I would not be surprised if it ran at a different molecular weight. IF you are seeing a dimer that is inhibited by a gamma-secretase inhibitor then I believe you are seeing the real NICD. Perhaps you see a dimer because we know that gamma-secretase can cleave at several locations and you can pick this up with this antibody. At this time I am uncertain as to the identity of the 23 kDa band. This antibody has only been tested in ELISA against the peptide immunogen, in which a strong response has been detected. This antibody has not been tested in other applications so I cannot really tell you what size band we would expect to see.

Can you please submit your results to fast-track@abcam.com so we can determine if the antibody is working properly?

I hope this information helps, please do not hesitate to contact us if you need any more advice or information.

Hi Jennifer,

Thank you for attending to this matter. We have had such good experiences with the AbCam reagents that I was surprised the last batch failed. I would like to try the newer batch you have available. Do you need any shipping information? We will test it out as soon as we receive it and I'll let you know the results.

Thank you once again,

ANSWER:

I'm sending you another vial of ab8926 as a replacement. For your record it is order# 48528 and you should receive it later this week or early next. Please do let me know how this batch works out for you. As I mentioned, ab8926 is a Fast Track antibody and thus has not been fully characterized and so your feedback is extremely valuable. Please let me know if you have any more questions.

Customer previously bought a vial of ab8926 back in the fall and it worked good in IHC-P. Recently bought another vial, lot# 46293 (order# 42237), and is getting very high background.

ANSWER:

Thank you very much for your patience. As you know, ab8926 is a Fast Track antibody and thus has not been fully characterized. We have not yet had any feedback regarding how this antibody works in applications other than ELISA, and so your feedback is very valuable. It is difficult to determine if the problems that you have experienced with the second batch were due to that specific lot, the antibody, or experimental conditions. We have a newer batch in stock and I can send you a vial of that batch to try. Please do let me know if you would like me to send that out to you.