Anti-Epac1 抗体 (ab21236)

Thank you for contacting Abcam earlier today.
The composition of antibody stabilization buffer has the following components: the exact concentrations of these constituents is proprietary in nature and cannot be disclosed.
Tris/Glycine buffer pH 7.4, glycerol, stabilizing proteins, cryogenic stabilizers, and preservatives (0.02% sodium azide)
This preparation cannot be used for coupling antibodies to other substrates,.
Please let me know if there is anything else I can help you with.

The rabbit monoclonal will be effective for IF. The application field lists ICC (iimunocytochemistry) which is IF (immunofluorescence) if one is staining cell cultures with a fluorochrome.

I have issued a free of charge replacement with ab109415.

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.

Thank you for your thorough reply. One other question I meant to ask was how much sample you load per lane of the gel. We recommend at least 20ug per lane, especially if no signal is evident.

Assuming you are loading enough sample, I suggest trying a different antibody as a free-of-charge replacement for ab21236. We do have another lot of ab21236 but there have been other reports of batch inconsistency and I think a rabbit monoclonal such as ab109415 will be a better choice for you. Please let me know if you would like to try this.

Click here (or use the following: https://www.abcam.com/Epac1-antibody-EPR1672-ab109415.html).

Thank you for bringing this to our attention. I am sorry to read that the antibody is giving you unexpected results in these two applications.

There may be a modification I can suggest to improve your result but it may be the case that, as you suspect, the antibody is defective. The other possibility, assuming your protocol is correct, is that the samples do not contain Epac1, or in amounts too low for the the antibody to detect. Do you have any other evidence, your own or from the litereature, that your cells contain Epac1?

For our records, could you please answer a few questions about your protocol?

Can you please tell me the species of the endothelial cells, and their origin? That is, is this a primary culture, or a continuous cell line?

Can you please tell me what you used for a blocking solution for the western blotting, and the antibody diluent?

Are you confident the transfer from gel to blot was successful? Finally, are you confident that the secondary antibody is effective?

If I cannot offer any suggestions to improve the protocol, and you are confident the cells express Epac1 protein, I think it will be best to replace the antibody, either with a vial from a different lot, if available, or a different Epac1 antibody. This replacement will be free-of -charge, assuming your samples are of one of the species listed on the datasheet (Human, Mouse, Rat and Rabbit).

I'm very sorry for the delay in replying to you. I have now received feedback from the laboratory that tested the antibody ab21236 and was informed that it was tested against recombinant Epac 1 and gave a very good strong band. However, it had poor affinity for epac 1 protein when it was tagged with a marker protein (Myc-tag). Similar results were obtained with His-tag. We believe this also depend upon the orientation of the tag marker. We are not aware of a suitable positive control of endogenous Epac1 but can provide in our catalogue the recombinant protein used to test this antibody. Please let me know if you would be interested and I will add this to the catalogue. We would recommend to try to immunoprecipitate the cell extract form 250ug-500ug total protein to enrich the sample and then do a western as I have been informed that this antibody does immunoprecipitate well. Furthermore, we recommend to incubate the primary antibody overnight at 4C and to boil the samples at 95C to make sure they are well denatured. Please make sure you are using reducing conditions in the loading buffer and that your protease inhibitors are fresh. Finally, the blocking buffer sometimes can alter the signal, it would therefore be good to try a different blocking buffer, milk or BSA (typically we use 5%BSA for 1 hour and incubate the antibodies in TBST only). I hope this information will help you, please do not hesitate to contact me if I can be of further assistance,