Asked 13th Nov, 2017

Is there a way to determine the quality of MUF labled substrates?

As a part of my MSc by Research we will be utilising high throughput microplate assays following Jackson et al. (2013), a technique that utilises 4-methylumbelliferone (MUF) labled substrates.
In our lab we have some of the necessary model substrates ( 4-MUF-β-D-glucopyranoside and 4-MUF-N-acetyl-β-D-glucosaminide) but their condition is unknown.
Does anybody know if there is a way to determine if they are still fit for purpose as they are expensive to purchase and it seems like a waste of money to purchase more if they are still good to use.
Thanks in advance for the help.

All Answers (3)

14th Nov, 2017
Adam B Shapiro
Entasis therapeutics
LC-MS could be used to study the purity and make sure the mass is correct. A lower-tech way would be TLC, but you would need a reference standard. If you look up the original synthetic paper for these substrates, it might give TLC conditions and Rfs, as well as a recrystallization method if repurification is needed.
17th Nov, 2017
Colin R. Jackson
University of Mississippi
If they've been kept reasonably carefully (dark/plastic jar, freezer or fridge as recommended by manufacturer) I've found that the substrates are a lot more stable than we think. Even if somewhat degraded, that degradation has likely released the MUF tag so you can detect that by measuring fluorescence of substrate solution. In terms of practicality, as long as your enzyme assays include substrate controls (the substrate alone - no sample) you can account for increased initial fluorescence of the substrate initially without major problems. I suppose you could have such serious degradation that your substrate concentrations are no longer in excess (so your reactions are substrate limited which brings up enzyme kinetics issues), but that's unlikely. You could buy purified b-glucosidase (etc.) and validate substrate quality by running assays using that pure enzyme, or just run a test set of samples (soil, water, litter) from anywhere. The b-glucosidase and NAGase you're assaying are common enzymes that you'll detect activity for in most environments.
22nd Nov, 2017
Lukas Kohl
University of Eastern Finland
The answers above are great, and I agree that your compounds are likely still good for use.
Still: Buy new ones.
The time you/your student work on establishing that they're still good will cost more that re-ordering the labelled substrates, and it's one less issue that could come up during review when you get your results published.

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