I'm using ubiquitin-AMC as the substrate for deubiquitination assays with several different deubiquitinases. Normally I'd do the assay in 50 mM HEPES (pH 7.5) and 150 mM NaCl, which I know works well. However I'm about to use two new deubiquitinases with estimated pI of 7.2 and 7.8 respectively, potentially crashing out at pH 7.5. I need to change the assay pH but every paper I find uses Ub-AMC at pH 7.5. Does anyone have any experience with it at alternative pH? I'm worried it won't be as active or the assay will be distorted
(NB: I know I could buffer samples at a range of pHs and check if they're in solution and if fluorescence is impacted, but reagents are running low and I don't trust supply chains right now)
Dear Elizabeth Radley,
The links below should help you out ...
https://southbaybio.com/products/ubiquitin-amc.html
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2447653/
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Here is some more relevant info:
What is the concentration of Ub-AMC in ubiquitin?
Concentrations of enzymes were fixed (UCH-L3: 100 pM; OTUD2: 10 nM; USP30: 10 nM) and concentrations of Ub-ACC and Ub-AMC substrates were set in the range from 50 nM to 10 μM.
Ubiquitin 7-amino-4-carbamoylmethylcoumarin as an improved ...
https://www.sciencedirect.com/science/article/abs/pii/S0040402016304409
Which is the best dub assay for ubiquitin 7A?
Among the DUB assay reagents, fluorogenic Ub-AMC (ubiquitin C-terminal 7-amino-4-methylcoumarin) 7a and Ub-Rhodamine 7b have been extensively used in activity profiling of different DUBs and inhibition screening of compound libraries in a high-throughput manner.
Ubiquitin 7-amino-4-carbamoylmethylcoumarin as an improved ...
https://www.sciencedirect.com/science/article/pii/S0040402016
Which is better ubiquitin or UB-ACC for dub?
A new fluorogenic substrate Ub-ACC (ubiquitin C-terminal 7-amino-4-carbamoylmethyl-coumarin) was developed for DUB (deubiquitinating enzyme) activity assays. This substrate can be synthesized with higher efficiency than the classical DUB substrate, Ub-AMC (ubiquitin C-terminal 7-amino-4-methylcoumarin).
Ubiquitin 7-amino-4-carbamoylmethylcoumarin as an improved ...
https://www.sciencedirect.com/science/article/abs/pii/S0040402016304409
Which is more sensitive to UB AMC or UB-ACC?
According to the DUB assays, Ub-ACC is nearly 2-fold more sensitive to Ub-AMC owing to the higher fluorescent yield of ACC. This permits reduction of the DUB enzyme and Ub-ACC concentration which may save costs for large-scale DUB inhibitor screening assays.
Ubiquitin 7-amino-4-carbamoylmethylcoumarin as an improved ...
https://www.sciencedirect.com/science/article/pii/S0040402016304409
Thanks Len Leonid Mizrah !
For reference for anyone reading this thread in the future with the same question - TL;DR AMC works between pH 3-11, and whilst it technically works outside of that fluorescence is significantly impaired.
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