Anti-ADAM17 antibody - Activation site (ab39163)

I have seen bands ~60kDa with ab39163, and from searching your website understand that these are due to n-terminal cleavage. What I wanted to know was whether or not it’s been shown exactly which portion of the protein this band represents

(ie, is it just the cleaved pro-domain, which contains the activation site epitope, and if so is the mw high due to glycosylation, or is it a different cleavage product?).

Thanks very much,

ANSWER:

Thank you very much for your inquiry.

The amino end of ADAM-17 is processed by furin-like proteinases during maturation of the protein. The Carboxy end is also cleaved by different proteinases, and the amino end is further cleaved as well.

The ab39163 ADAM-17 epitope is after the furin cleavage site, within the range of the catalytic domain.

Most of the glycosylation occurs in the propeptide domain, so a larger than expected size might be ADAM-17 with intact carboxyterminus.

This has not been experimentally tested.

I hope this information is helpful. Please do not hesitate to contact me again with any further questions.

I have been using your rabbit polyclonal against the propeptide domain of ADAM17 to examine an ADAM17 prep and get no recognition with it at all whereas your other Abs (ab39163 and ab28233) are fine. I think my protein has been cleaved following Arg58 in the propeptide domain and I suspect that your Ab has been made against a peptide within the missing N-terminal 58 amino acids. Is this correct? Also, for the activation site Ab (ab39163) is only the intact site recognised or if after cleavage are both sides recognised or only one?

ANSWER:

you are correct in assuming that ab39161 recognizes only the prodomain cleaved during ADAM17 maturation. This maturation can be prevented using divalent metal chelators such as EDTA or EGTA. One of the customers who submitted an Abreview for ab39163 noted that "Seems to be selective for Precursor forms of ADAM17". The presence of the 55kDa band as well would indicate that both the cleaved and uncleaved form of this protein are recognized.

I have another question with your antibody ab39163, on your data sheet said "WB: Recommended starting dilution of 1/1000 (when using colorimetric substrates such as BCIP/NBT) and 1/5000 (for chemiluminescent substrates). Higher concentrations of antibody may be needed for samples from more distantly related species. Detects a band of approximately 120 kDa in reduced Western blots of conditioned media or cell lysates, which is converted to a 55-60 kDa band. (Predicted molecular weight: 93 kDa). Note: EDTA/EGTA treatment of tissues or lysates is required to see latent zymogen. Dilution optimised using Chromogenic detection. Not yet tested in other applications. Optimal dilutions/concentrations should be determined by the end user.". Why can 120kD become 55-60 kDa ?

ANSWER:

Thank you for your enquiry.

Regarding the difference in detected molecular weight, glycosylation and other post-translational modifications increase the apparent molecular weight of ADAM 17 to 120 kDa, and cleavage at the aminoterminal end results in lower molecular weight forms, therefore producing the 55-60 kDa range.

If there is anything else I can help you with, please do not hesitate to contact me.