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I recently ordered an anti-tetherin antibody (ab88523) and tried it in a western blot last week. There are a number of non-specific bands and no band at the size stated in the datasheet. There is a band at lower size but I am not sure if this is correct. The cell lines used were Vero and two types of Hela cells, the Hela particularly should express large amounts of tetherin. I used the antibody at 1 in 200 overnight at 4 degrees. I have attached a picture of the blot and any advice you could give me would be appreciated.
Asked on Mar 12 2012
Thank you for contacting us and letting us know about the trouble with this antibody.
I do have a couple of additional questions, so that I can better understand the situation:
1) Do you know your order number or lot number?
2) How has the antibody been stored?
3) What solution do you use for blocking the membrane (BSA or milk)?
4) How were the lysates prepared (reduced, denatured, protease inhibitors used)?
5) How much protein was loaded onto the gel per lane?
6) On the image that you sent, what are the top two molecular weight markers? I'm not sure if these are 40 and 50 kDa?
7) On the image, what is the difference between the lanes? Are these different lysates, and if so which is in each lane?
8) Were any of the cells treated in order to induce tetherin expression, or are you analyzing endogenous levels?
According to SwissProt, the tetherin primary amino acid sequence is cleaved to produce the mature protein, which removes 19 amino acids. This mature protein corresponds to about 18 kDa, which is close to the lower band on the image. However this mature protein is generally glycosylated and is seen by WB around 30 kDa using ab88523. At this point I'm not sure if the lower band on the image corresponds to tetherin.
We do guarantee all of our products to work as stated on the datasheet for up to 6 months after purchase, so I would be happy to send a replacement or alternatively issue a credit or refund. We also have ab14694, which as been cited in two references-
I look forward to hearing from you. Please let me know if you have any questions or if there is anything else that I can do for you.
Answered on Mar 12 2012