Anti-BST2 antibody (ab88523)

Yes, this is the correct order. I would be grateful if you could send the other antibody and I will try it straight away.
Many thanks for your help.

ANSWER:

Thank you for your reply.
I'm sending a free of charge vial of ab14694 on the order ***, which should arrive on Monday. Please keep me updated about the performance of this replacement antibody.
I look forward to hearing from you. Please let me know if there is anything else you need from me.

Good morning,
Apologies for my delay in replying!!
In answer to your questions:
1. Lot number ***
2. Antibody in stored at -20 and was used within a week of delivery
3. Milk
4. Denatured
5. Approximately 10ug
6. Yes they are 40 and 50kDa
7. Yes they are different lysates
Lane 1. Vero cells
Lane 2. Hela cells
Lane 3. Hela cells (a new batch)
We suspect the Hela will have more BST-2 than the Vero, certainly Hela do express BST-2 according to the literature.
8. All endogenous levels
I am happy to try the other antibody you suggest with our lysates, if we get a refund on this one of course!!
Many thanks for your help,

ANSWER:

Thank you for your reply and for providing so many details about the protocol. This information will be helpful for our investigation of this antibody.
I've found an order going to ***. Can you confirm that this is your order? I can send the replacement antibody, ab14694, free of charge if you would like to try it, and if it still does not work then we would be happy to issue a credit or refund.
I look forward to hearing from you and resolving this promptly. Please let me know if you have any questions or if there is anything else that we can do for you.

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.

ANSWER:

According to our records, ab88523 was proving difficult to use in Western blot and we were in contact in order to help resolve the issue.
Looking at our correspondence, it appears that we are awaiting more details in order to help us better understand the difficulties experienced. If the requested information has already been sent, it appears that it did not reach our Scientific Support team and we apologize for this inconvenience. In this case we would like to ask for the information again so that we can reach a resolution.
If the issue has already been settled, please let us know so that we can be assured that the problem has been solved to your satisfaction and update our records.
We wish you the best of luck with your research and look forward to a reply.

Good morning,

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.

Regards,

ANSWER:

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-
http://www.abcam.com/BST2-antibody-ab14694.html
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.