Anti-Leptin Receptor抗体(ab5593)

Thank you very much for keeping me updated about the situation, and I'm sorry to hear that the new lot is also not giving the expected results.

As mentioned before, your protocol looks very similar to the protocol used by the lab to test the antibody (attached).In our testing, the 125 kDa band looks fainter than the 100 kDa band, butthe Western blot images in the literature do not show the 100 kDa band so I can not comment on their results.I've reviewed all of the information that I could find on this antibody, and it seemsthat the antibody has been tested with the membrane fractions of the lung and brain lysates (including in our lab testing), so I would suggest trying a membrne fractionation and seeing if this enriches the samples enough to visualize the 125 kDa band. We have a subcellular fraction protocol that could be used-

https://www.abcam.com/index.html?pageconfig=resource&rid=11473

As this protein is heavily glycosylated and also at a higher molecular weight (meaning difficult to transfer), I'd also suggest running a Ponceau stain on the membraneto make sure that the long isoform efficiently transferred.

I'm also wondering if the tissue lysates are whole brain and lung, or are they specific regions of these organs? I can check with the lab whether their samples were taken from the same region.

I look forward to hearing from you. I am very sorry for all of the trouble with this antibody, but I hope to resolve this as quickly as possible. Please let me know if there is anything else that we can do for you.

Thank you very much for participating in our recent survey.

I am very sorry that the long form of the OBR protein is still not being picked up with this antibody. Have you tried the membrane fractionation to enrich your samples, or the Ponceau stain to verify protein transfer (mentioned in my previous email)? Are you using whole lung and brain lysates, or lysates from specific regions?

We do guarantee these results and I can send a replacement antibody or issue a credit or refund for your original purchase if you would like. Please keep me updated with any new results and let me know if there is anything else that we can do for you.

Thank you very much for your reply.

I'm sending a free of charge vial from a new lot on the order ***, which should arrive tomorrow.

Please keep me updated about the results with this new vial, and if there is anything else that we can do for you please let me know. I'll be happy to help.

Have a nice day!

Thank you very much for your reply, and I apologize for the trouble with this antibody.

I look forward to hearing from you once you have discussed the options with your supervisor. Please let me know if there is anything else that we can do for you.

Have a nice day.

Thank you very much for your emails and for your patience.

I have been discussing the situation with my colleagues in the lab, and I've found out that the mouse lung lysate used for the WB on our datasheet also came from a healthy, normal-fedmouse.According to the literature (as you have also found), the long form of this receptor is not expressed in obese or diabetic mice, but is expressed in wild type C57Bl/6J mice, so we are surprised that the receptor is not detected in these samples. One of my colleagues has found in the literature, though we have not tested, that mRNA of the long form of the receptor increases during fasting, so this may be one way to boost expression and detection of the 125kD product.Additionally, we loaded 40ug of lysate per lane for detection of both bands in the WB image on the website, which you have also loaded as well. The rest ofour protocol is also very similar to yours, so we are not sure exactly why the long form is not being picked up in your samples.

We do fully guarantee this antibody to work as shown on the datasheet, so I would be happy to send you a free of charge replacement vial from a different lot in order to compare the results. Would you be interested in this option? Alternatively we can issue a credit or refund for your purchase.

I look forward to hearing from you. Please let me know if you have any questions or if there is anything else that we can do for you.