Question (31895) | Anti-Proteasome 20S LMP7 antibody (ab3329)

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Question

Dear Madam/Sir, We have recently purchased an antibody (20s LMP7) #ab3329 Lot no. GR39959-4, from Abcam but it does not seem to be functional. Me and my colleague have tried it with both milk and BSA blocking solution and it yields two non-specific bands much higher and lower than expected (20kDa) size. We also have purchase the Smurf1 and Smurf2 antibodies. The Smurf1 antibody works good but Smurf2 (#ab38543) antibody weakly gives the expected band but it shows also lots of sand shaped spots on the membrane and washing for long time does not solve this problem. I wonder to know if we could get replacement for these two new purchases from another batch of antibody. Thank you for considering our case. Our shipping address is

Answer

Thank you for taking time to send us your protocols. I am sorry to hear that this antibody is not providing satisfactory results. The details provided will enable us to investigate this case and will provide us with vital information for monitoring product quality. Having reviewed this case, I would like to offer some suggestions to help optimize the results from ab38543. Spotty/sandy looking blots are often the result of aggregates in the block or antibody. I would recommend when preparing the milk in TBST that you stir it with a stir bar for quite some time. This will help dissolve particulate matter that can stick to your blot. Filtering the milk will also remove any milk that has not dissolved. I would also suggest that you centrifuge the antibody before use and use the supernatant from the top should aggregates appear. You may use the supernatant at recommended dilutions without a reduction in quality. I would also recommend using a lower concentration for your secondary antibody. Some people have noted that rabbit secondaries used at high concentration may cause issues like this. To increase target signal, I would suggest using a higher concentration of ant- SMURF 2 antibody. This product has been used successfully at concentrations as high as 1:50. 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. In regards to ab3329. I've seen that others have posted abreviews which have shown a higher non-specific band at about 66 kDa. Is this about the range of the higher band that you have reported? Do you have an image that you might send us? Would it also be possible to send me the original order number or purchase order for these products. If we need to replace them having that information will expedite the process. I hope this information is helpful, and I thank you for your cooperation.

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