Anti-APC antibody [T45] (ab45764)

If you could arrange for the alternate antibody to be sent that would be ideal,    

ANSWER:

As requested, I have issued a free of charge replacement with the order number 996071 (one unit of ab15270).

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.

I have altered the method as you suggested. I typically transfer at 10V overnight (as per the instruction manual from BioRad) so I did this with a 50V boost for 30mins. I obtained an even stronger signal with my other antibodies, however there was no bands for the APC antibody, even after a 2 hour exposure. Are there other abcam antibodies that react with Mouse that I might be able to get as a replacement? regards  

ANSWER:

Thank you for your message and for providing this further information.

I am sorry to hear the suggestions made have not improved the results on this occasion. I appreciate the time you have spent on these experiments and would be pleased to arrange a replacement, a refund or credit note in compensation. The alternative antibody we have and that has been tested in western blot and mouse is ab15270.

I look forward to hearing from you with details of how you would like to proceed.

I purchased the Anti-APC antibody [T45] (ab45764).

However, after two western blots, I have had no success in detecting APC from samples that should definitely express the protein – extracts from mouse intestinal epithelium. I’ve included a brief outline of the protocol used at the bottoms of this email. It is normally very reliable (including with other AbCam antibodies) however if you believe there might be a problem with the method please do let me know.

I cut the membranes to blot for smaller proteins (raptor 150kDa, actin 42kDa and rps6 32kDa) and did detect signals for these proteins using other antibodies. The Rat secondary used was a donation from another lab that has not had any difficulty using it to detect other rat-primaries. This leads me to believe that it is the primary antibody that is not performing as advertised.

If you could send me a replacement antibody that is reactive with Mouse, that would be much appreciated

regards

Method 20ug total protein/lane. Samples from mouse intestinal homogenate. 8% acrylamide gel Ran such that 250kDa marker was well within the resolving gel, so that an proteins ~300kDa should be detectable. wet transfer (50 mM Tris, 192 mM Glycine, 20 % methanol) 4oC overnight

Ponceau staining showed good transfer, including for high MW proteins. Blocked in 5%Milk-TBST, 1hr, room T. APC antibody 1:1000 in milk-TBST, 4oC, overnight with rotation. washed 3times with TBST and incubated RT,1 hr, with anti-rat secondary (sigma) washed 3times and covered with ECL Plus for 5mins. Exposed for 5, 10 and 45 mins.

ANSWER:

Thank you for contacting us. I am sorry to hear that this antibody is not providing satisfactory results.

Western blots of big proteins such as APC can be difficult. I looked at your protocol and I think some changes may help to solve the problems you are experiencing.

- When testing our antibodies, our lab uses 5% BSA as a blocking reagent, so I recommend switching to this instead of milk. Some antibodies give stronger, more specific signals on blots blocked with BSA instead of milk, so doing this may improve the results you are seeing, and reduce the non-specific bands. An example of the above is the western blot obtained with the Abcam GAPDH antibody ab9385 : Click here for the western blot image using ab9385 (or use the following: www.abcam.com/ab9385).

- I would highly suggest to optimize the transfer conditions by increasing the transfer time to 24 hours (in some examples, for big proteins, the transfer is done for 48 hours)

- A transfer at 30V for a long period of time, like you did, is fine but it has been shown that a "boost" at 70-80V for 1 hour at the end of the transfer can be of significant improvement.

- You can also use up to 0.05% SDS in the transfer buffer, and reduce the proportion of methanol to 10%. The reason is that methanol washes out the SDS from the protein.

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.