Anti-Notch1 antibody - Cleaved - Val1744 (ab52301)

Thanks for support and the advice. We retested this antibody again, followed that from your suggestions. We used the citrate buffer (at PH6.0) for the antigen retrieval, incubation time increased to 25 minutes, which the previous retrieval was a 16 minutes. The secondary antibody should have no problem as we used this antibody for quite some included some tests from abcam primary antibodies. They are all for immunohistochemical based observations.

We are unfortunately unable to observe optimal result this time.

So, we would like to try if you would send us a replacement lot.

ANSWER:

Thank you for your reply.

My colleague is out of the office today but I will be happy to help you.

I'm sending a free of charge vial of ab52301 from a new lot, which will arrive on order ***. This order will be shipped shortly and should arrive early next week.

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

We have a technical inquiry as shown attached. Looking forward to your advice or suggestions.

ANSWER:

Thank you for contacting us. I am sorry that ab52301 is giving no signal in IHC. The protocol you provided looks fine, but could you please clarify what antigen retrieval buffer was used (what pH) and how long was it incubated? What secondary antibody was used and at what dilution? Increasing the duration of the antigen retrieval step or the concentration of the secondary may help to increase the detected signal.

I appreciate the amount of troubleshooting you have done. If you are unsatisfied with your results, I would be happy to offer you a free of charge replacement or credit. Please let me know the original order number and how you would like to proceed and I will be happy to assist you further.

Dear Tech support One of our customers is looking for an anti Notch1 antibody that recognizes only the trans membrane portion of the protein. The customer wishes to measure the quantity of only the transmembrane part of Notch1. I have searched a proper antibody in Abcam's results but was unable to find a trans membrane specific antibody. Is there a suitable antibody available? Thank you for your kind help. Regards,

ANSWER:

Thank you for contacting us. We have a number of anti-Notch1 antibodies in our catalogue, however, none are specifically raised to detect the transmembrane domain. The transmembrane domain of human Notch1 is composed of residues 1736-1756 (SwissProt: P46531). We havean antibody which have been raised against an immunogen taken from residues 1755-1767, just outside the region of interest (http://www.abcam.com/activated-Notch1-antibody-ab8925.html). We also have an antibody which has been raised to specifically detect the cleavege site betweenglycine 1753 and valine 1754 (http://www.abcam.com/Notch1-antibody-Cleaved-Val1744-ab52301.html). This binds to the C-terminal cleavage product produced from this andit only recognises the cleaved form. Unfortunately these are the only antibodies I am able to suggest to your customer. I am sorry that I have not been of more help.

LOT NUMBER GR15878 ORDER NUMBER -- NOT SPECIFIED -- DESCRIPTION OF THE PROBLEM I observe multiple light bands. SAMPLE mouse kidneys PRIMARY ANTIBODY The primary antibody concentration was 1/250. The incubation was done overnight at 4 degrees followed by 3 washes (one 15 and 2 5 mins washes) DETECTION METHOD Chemilumescent substrate POSITIVE AND NEGATIVE CONTROLS USED Did not have a positive control. Requesting abcam to send a vial of cell lysate that can be used as positive control ANTIBODY STORAGE CONDITIONS Stored the antibody at 4 degrees SAMPLE PREPARATION Homogenized the tissue in RIPA buffer AMOUNT OF PROTEIN LOADED 50ug ELECTROPHORESIS/GEL CONDITIONS Gel was run in reducing conditions in a 4-12% Bis-Tris gel TRANSFER AND BLOCKING CONDITIONS The trasfer was a wet trasfer using Invitrogen NUPAGE transfer buffer onto nitrocellulose membranes. The blocking was done in 5% milk in TBS SECONDARY ANTIBODY THe secondary antibody used was ab6271 ( goat anti-mouse HRP). The incubation was done for 1 hour at RT. Incubation was followed by 3 washes ( one 15 and 2 min washes) HOW MANY TIMES HAVE YOU TRIED THE APPLICATION? 1 HAVE YOU RUN A "NO PRIMARY" CONTROL? No ADDITIONAL NOTES Since the antibody binds to intracellular doamin of NOTCH1, do you get 2 bands ( one at ~250kDa and another at 90KDa)? Also could you please send me a vial of cell lysate that can be used as a positive control.

ANSWER:

Thank you for contacting Abcam. I have talked to the lab about whether ab52301 recognizes other bands. They have told me that the antibody can detect endogenous levels of fragment of activated Notch 1 resulting from cleavage adjacent to Val1754. The band at 120kD is the cleaved form, and the band at about 270kD is the full length form. Sometimes it can detect the full length form. The band that you are seeing at around 100kDa could be further breakdown products of your protein of interest or non-specific binding of the antibody. Also looking at some of our other Notch-1 antibodies (I am looking at ab65297), they also detect a (faint) band at ~100kDa in human kidney samples (in this case HEK cells). I have searched out catalogue for any lysates that you could use as a positive control but I have not found anything suitable. Please let me know if there is anything else I can help you with.

LOT NUMBER GR15878 ORDER NUMBER -- NOT SPECIFIED -- DESCRIPTION OF THE PROBLEM I observe multiple light bands. SAMPLE mouse kidneys PRIMARY ANTIBODY The primary antibody concentration was 1/250. The incubation was done overnight at 4 degrees followed by 3 washes (one 15 and 2 5 mins washes) DETECTION METHOD Chemilumescent substrate POSITIVE AND NEGATIVE CONTROLS USED Did not have a positive control. Requesting abcam to send a vial of cell lysate that can be used as positive control ANTIBODY STORAGE CONDITIONS Stored the antibody at 4 degrees SAMPLE PREPARATION Homogenized the tissue in RIPA buffer AMOUNT OF PROTEIN LOADED 50ug ELECTROPHORESIS/GEL CONDITIONS Gel was run in reducing conditions in a 4-12% Bis-Tris gel TRANSFER AND BLOCKING CONDITIONS The trasfer was a wet trasfer using Invitrogen NUPAGE transfer buffer onto nitrocellulose membranes. The blocking was done in 5% milk in TBS SECONDARY ANTIBODY THe secondary antibody used was ab6271 ( goat anti-mouse HRP). The incubation was done for 1 hour at RT. Incubation was followed by 3 washes ( one 15 and 2 min washes) HOW MANY TIMES HAVE YOU TRIED THE APPLICATION? 1 HAVE YOU RUN A "NO PRIMARY" CONTROL? No ADDITIONAL NOTES Since the antibody binds to intracellular doamin of NOTCH1, do you get 2 bands ( one at ~250kDa and another at 90KDa)? Also could you please send me a vial of cell lysate that can be used as a positive control.

ANSWER:

Thank you for contacting Abcam. I am sorry that you are experiencing issues with ab52301 in western blot. The antibody is covered under our Abpromise which means that it is guaranteed to work in western blot on mouse samples. If we cannot resolve the problems of multiple bands, then I would be happy to find a suitable replacement or process a refund. I was looking through your protocol and you mention that there are multiple faint bands. What sizes are those bands at? Do any of the observed bands appear at either 250kDa or 95kDa? The data we have on the antibody shows that it does detect a band at approximately 95kDa. If some of these faint bands are not at the predicted sizes, then you could lower the primary antibody concentration to 1/500, which is the antibody dilution that was used when testing the antibody. Using too high a concentration of primary antibody can cause non-specific binding to occur. As for a positive control, the kidneys do express a high level of Notch1, however if you are concerned then next time you prepare your sample, you could also prepare some tissue from the GI tract, which also expresses high levels of Notch1. I look forward to your reply.