Anti-Hephaestin antibody (ab56729)

Dear ,
i submitted the Abreview as promised.
Please delete the earlier version (without the discount code)that I
submitted without intention.
Can I order now the free antibody?
Thanks

ANSWER:

Thank you for your reply.

Your Abreview has successfully been received and one of our colleagues will review it in order to publish your results in our Website.

The Abreview discount code is now activated; you can use it to place a new order for one primary antibody completely free. Please do not forget to mention the discount code in your order.

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thanks for the message.
The problem with the higher MW band over the 160 KDa marker (about 170
KDa) is that it can only be seen with a very prolonged incubation with ECL
advance, which makes appearing a whole panel of aspecific proteins.
Given that HEPH is highly expressed on muscle, isn't that strange to get
such a faint band?
I found on Human Atlas a variety of MW from 90 to 130 KDa, although not as
few as 65KDa http://www.proteinatlas.org/ENSG00000089472/antibody
I can try another blot according to your protocol and come back to you.
I really hope we can quickly find a solution

ANSWER:

Thank you for your reply.

If you do not detect the correct band after adjusting your protocol, please let me know and I will either replace or refund the antibody for you.

In the meantime if there is anything else I can help you with, please let me know.

I did some research and checked all the WB we did with anti HEPHAESTIN
ab56729.
The protein is known to be abudantly expressed on human skeletal muscle,
therefore a good signal is expected.
I hereby attach a ppt showing the results. Only a band at around 65 KDa
could be clearly and consistenly seen on human skeletal muscle (slide 1).
Only using ECL advance, several higher MW bands can be seen but none at
the expected MW of 130 Kda.
Conditions: I Ab 1: 500 in milk 5% TTBS 1h RT, 4 wash in TTBS, II Ab
1:5000 in milk 5% TTBS, 3 wash in TTBS.
Do you think that the very clear band at 65 KDa could be really
Hephaestin? That is approximately half of the expected MW!
Other suggestions?
If you need the complete protocol infos or other informations please let
me know.
I urgently need to make this antibody work.
Thank you very much,

ANSWER:

Thank you for your reply.

I do not think that the 65kDa band is Hephaestin, it could be that the antibody is recognizing something non-specifically on skeletal muscle tissue.

However there does seem to be a band at a higher molecular weight (˜160kDa) Hephaestin? I say this as according to the http://www.uniprot.org/uniprot/Q9BQS7 sequence, there are a number of glycosylation sites on this protein, which could explain the slightly larger sixe than expected.

To reduce the non-specific binding you are seeing and to check whether the 160kDa band is the correct target, I would alter you protocol slightly:

1 - Block in 5%BSA rather than milk.

2 - Use a 1/1000 primary antibody concentration, with overnight incubation at 4C.

The antibody is covered under our Abpromise for six months and is guaranteed to work in western blot on human samples. If we cannot resolve the issue you are having with the antibody then I would be happy to either send a replacement antibody or to process a refund.

Please let me know if there is anything else that I can help you with.

thanks for the message. I am sending you the WB on monday.
Do you know which could be a reliable source/database to make sure the
protein is expressed in skeletal muscle?

ANSWER:

Thank you for contacting Abcam.

You could use http://www.proteinatlas.org/ to check for expression levels in human otherwise you should consult the literature and see what is published about the expression pattern of this protein.

Please let me know if there is anything else I can help you with.

I have purchased AC 56729 for Western blotting in human muscle.
However I cannot see any band at 130 KDa, but multiple bands at or below
110 KDa.
I would kindly ask you:
-Are you aware if this protein is expressed in human muscle?
-If yes, which could be the problem in this case?
Thanks,

ANSWER:

Thank you for contacting Abcam.

For ab56729, the Anti-Hephaestin antibody from what I can see this protein is detected in breast, colon, bone trabecular cells and fibroblasts. I am not completely familiar with the protein distribution pattern of this antibody but if you feel that the protein is expressed in muscle tissue then I would be more than happy to help determine why you are not seeing a signal in western blot. If you can send me a copy of the western blot protocol that you are using, I would be more than happy to see if there are any protocol variations that I can suggest.

If there is anything else that I can help you with, please let me know.