Anti-AUTS2 抗体 (ab96326)

Unfortunately this is the only buffer formulation available for this product at present. You may remove the glycerol by use of an Antibody Concentration Kit (ab102776). Dilute the antibody to reduce the glycerol’s concentration, which in turn will make the buffer less viscous and easier to spin out. You can then use the spin column to concentrate the antibody back to its original concentration and volume, after having performed the buffer exchange.

Thank you for your reply and for providing the protocol information.

Your protocol looks ok to me and so should be fine.

I just had a few other questions, have you done a no primary control? Also now that I look at your blot again, you are getting the exact same bands in each lane, the main difference is that the ˜100kDa band in the WT lanes (on either end) are slightly more intense, yet the beta tubulin signal in these lanes is slightly lower. Is the results you were expecting?

I was just looking at the mouse sequence for this protein on UniProt (http://www.uniprot.org/uniprot/Q8VDM3) and the predicted molecular weight for the this is ˜90kDa, which is closer to the size your seeing. But we have other western blot data on our webpage from other customers who got a band at 139kDa in mouse tissue. So, I will be honest and say I am a little confused about what size of band you should expect to get.

If you have not already done so, I would suggest running a no primary control, to see if there is any non-specific binding from the secondary. I would also suggest loading as much protein sample as you can into each well and then using the primary antibody at 1/500 with overnight incubation at 4C, to see if there is any hint of a band at 130-139kDa. If after that, we still have not been able to resolve this issue, then I would be happy to refund the cost of the antibody.

If there is anything else i can help you with, please let me know.

Thank you for contacting Abcam.

The antibody, ab25788, is covered under our Abpromise for six months and is guaranteed to work in western blot on mouse and human samples. We have not yet tested the antibody in IHC and therefore it may not be suitable for that application, which would explain why you are not seeing the expected staining pattern. But as the antibody is guaranteed to work in western blot if we cannot resolve the issue you are having with the antibody then I would be happy to either send a replacement antibody or to process a refund.

To be able to better assist you in this matter, would you be able to send me a copy of the protocol that you have been using with ab96326 and I will see if there are any protocol suggestions that I can make.

I look forward to your reply and helping you resolve this issue.

Thank you for that information.


Can I just clarify something. You mentioned that the gel was probed twice, onceusing 1% milk in 1x PBS 0.3%Tween.This was then restained using5% milk in 1xTBS 0.05%Tween. For the first condition did you see any bands in any of the wells?If you could send the imageobtained would be very helpful.


Did you clone the vector used yourself? Do you have the full sequence (with the AUTS2 sequence included) which I could look at?


Could I also ask what transfer buffer you are currently using? Larger proteins sometimes precipitate in the gel hindering the transfer process. This can be reduced by adding SDS to a final concentration of 0.1% in the transfer buffer. Reducing the methanol content of the buffer to 10% can also reduce this phenomenon and aid in the transfer. As you are using a PVDF membrane you can remove methanol completely from the transfer buffer.


The only other thing I couldsuggest from having reviewedyour protocol isyour lysisprocedure.You may achievemove fulllysis by incubating with theRIPA for a little longer (we typically recommend 30 minuteson ice with agitation)andpellet thesample fully bycentrifugingat 16,000 x g for20 minutes at 4°C.


From the gel you have shared with me and the conditions used, we should be seeing something with the 293T cells. As the antibody is able to pick out the mouse AUTS2 quite clearly from the mouse whole brain lysate it may be that the protein is not concentrated enough to be seen. What I would initially suggestis that you reduce the amount of mouse whole brain loaded (to ˜10 fold less than currently used) and I would increase the amount of protein loaded for the Neuro2A and 293T cells. Initially I would just concentrate on the untransfected 293T and overexpressed/transfected 293T. Of these I would load a range of concentrations of protein: 30, 60, 90 and 120 µg/mL. If the AUTS2 protein is present then it should be detected under these conditions. Incubating with the antibody overnight at 4°C can often also improve the signal so I would suggest you do this.


If possible, I would also try initially reducing the blocking conditions (block as usual but dilute the antibody in only 0.1% milk with TBST). It is often also worthwhile to try blocking with BSA rather than milk. For unknown reasons some antibodies give stronger, more specific signals under BSA blocking, an example of this can be found from the following link:


https://www.abcam.com/gapdh-antibody-hrp-loading-control-ab9385.html


I hope these suggestions give improved result.If they do not then please do let me know. Finally, in my investigations I came across the following paper whichmay be of interest to you (if you haven't already come across it):


http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2818569/pdf/nihms162026.pdf

Thank you for calling us and for alerting us to the problem you are experiencing with Anti-AUTS2 antibodyab96326. 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 ama questionnaire so thatI can gather further information regarding the samples tested and the protocol used. This will hopefully allow me to more fully understand why the expected band is not being observed. This information will also allow us to investigate this case internally and initiate additional testing where necessary.


In the meantime, I will also look into the isoforms which can be expected to be detected by this antibody.


I look forward to receiving your reply.

Thank you for contacting us. The immunogen that was used to create ab96326 shows 100% identity with human long isoform of AUTS2 and 89% identity with short isoform of AUTS2. So this antibody will be detecting both the isoforms. I hope this information is helpful to you. Please do not hesitate to contact us if you need any more advice or information.

Thank you for contacting us. I have read the whole protocol you have kindly submitted. In order to address the problem quickly I have further questions to ask. - According to the attached image you have used non transfected N2a mouse cells, Lymphocytes supernatant and Lymphocytes pellet as a negative control right? I am sorry I am surprised by the fact that 1st and 3rd (very very faint band may due to spillage from 4th lane) there is no band while there is dark band in 2nd lane. Could you confirm there was no intermixing of lysates? - What is the band size you are expecting to be? - The description of the problem also does not correlate with the image. Does this mean the Lymphocyte pellet has HA-tag inserted cells? I predict from the image; the problem only is that antibody is detecting a band in Lymphocyte supernatant which it should not do. Right? - Could you specify which protein you have tagged with HA? Have you done any western blot studies with anti protein antibody? - Could you provide the order details so that I check if the delivery was on time? I will look forward to hearing from you soon.

Thank you for your email. I can confirm that I added the discount code to our internal documents of the Abreview 26773. Your customer can now redeem this discount code and order a free antibody. Please thank your customer for her valuable feedback and wish her ongoing good luck with her research.

Thank you for your recent enquiry which has been forwarded to me as my colleague is currently away from the office. I can confirm that ab96326 Anti-AUTS2 antibody was used to detect endogenous AUTS2 in 293T and NIH3T3 cells in the images on the datasheet. Revieiwng other antibodies on the internet for this target protein, many of them are tested in 293T cells. I hope this will provide some reassurance and is helpful. If you have any further questions, please do not hesitate to contact us.