Anti-Adenosine A1 Receptor抗体(ab3460)

Following up my previous message, the immunogen for ab124780, an alternative to ab3460, is also unfortunately unlikely to react with canine Adenosine A1 Receptor. Alignment of the canine receptor amino acid sequence with that of the ab124780 immunogen returns 60% identity. We generally consider this to indicate a low probability of cross-reaction.

Please let me know if we can help in any way.

Thank you for contacting us. I think the issue is probably that the homology between the immunogen and the canine receptor is low: 67% identity. We do not have a dog AAR1 to test this.

In general, for multi-pass membrane receptors, we suggest not boiling the sample before loading into the gel , to discourage the aggregation that boiling can induce. Heating at 70C for 10 minutes should be sufficient to denature the sample. (Aggregates do not enter the gel easily). Otherwise, the protocol should be standard, blocking with 5% milk or BSA, and incubation with the antibody diluted to 1/500 in TBST and incubating overnight at 4C. We suggest loading 20-50ug of sample per lane.

Have you considered any of the other AAR1 antibodies we have? None have been tested for reactivity with dog but I will check the immunogen for ab124780 against the dog sequence (http://www.uniprot.org/uniprot/P11616.fasta) and will send the result of the alignment when I have it.

Thank you for your reply. If the signal is stronger on frozen section compared to formalin-fixed sections, it indicates that the epitope unmasking may not be proper. We would suggest trying to treat the samples in microwave for different times and to see if the intensity of the signal improves. It is know that certain tissue requires longer retrieval than the other and even if you use a standard well characterized protocol, it may be worth optimizing the technique. As you have suggested, it may well be that the expression level is lower in the lung. One comment on the RT-PCR results - you may need to handle RT-PCR data very carefully. My experience is that high RNA levels do not always correspond with high levels of transcribed protein. If you need anything further or any help then please let us know.

Thank you for your enquiry and for your patience. We are very sorry to hear that you are having problem with this antibody. We have searched our database and found that this is a popular selling product and your feedback is the first we have received about it not working. Therefore, at this stage, we would suggest that there is either a problem with the vial you received, or modifications to your protocol are needed to obtain a positive result. A1AR protein is highly expressed in brain (especially cerebellum, hippocampus, thalamus, and cortex) and spinal cord and in part, modulates neurotransmitter release. This antibody was tested in human brain (hippocampus sections) and the pattern is plasma membrane staining. Therefore we would suggest using positive control (i.e. human or rat brain sections) along with the samples. If you use formalin-fixed sections we would suggest antigen retrieval (microwave method), but please make sure that the samples are not over-treated. We would also suggest blocking endogenous peroxidase activity which may give unspecific staining. If you are still having problem with this antibody, please contact us again providing the batch number.