I performed activity assays on cell lysates following the protocol described in Osellame et al. 2013.
I purchased 4-Methylumbelliferyl β-D-glucopyranoside from Sigma (M3633) and in the datasheed it is described as soluble in N,N-dimethylformamide
(DMF, 50 mg/ml), but for enzyme assays, it suggests to dissolve it at
5 mM (1.7 mg/ml) in water or 0.2 M sodium citratephosphate
buffer. I tried the second option and I couldn't get a fully solubilized solution (some white particles were still present in suspension in the buffer) which affected the compound concentration.
Any idea about why I am getting this problem and how to solve it? I have to say that this problem occurs randomly and is not associated to a specific batch of 4-Methylumbelliferyl β-D-glucopyranoside or buffer I am using.
Thanks for any help.
Ciao Nicoletta,
I don't have experience in this but I think that you can change slightly the pH of the buffer used to solubilize it.
You might want to incubate your 4-Methylumbelliferyl β-D-glucopyranoside in 0.2 M sodium citrate buffer at 42°C (water bath) for 5-10 min. When I worked with this reagent it usually helped to dissolve it completely.
Dear Nicoletta,
i suggest that you dissolve 4-Methylumbelliferyl β-D-glucopyranoside in water to make 5mM solution. Since the optimal pH for this enzyme is 5, you might adjust the pH of compound solution to 5.
Use fresh solutions for the enzyme activity.
Please note, that upon use, stored buffer or aqueous solutions might result in precipitation of the tested compound. In addition, since your compound is lactone it might be hydrolyzed in the presence of acidic or basic aqueous solutions. Therefore, 4-Methylumbelliferyl β-D-glucopyranoside aqueous solutions should be freshly prepared and used immediately (avoid any lengthy storage of the solutions).
Hoping this will be helpful,
Rafik
Thank you everyone for all the suggestions! I will try and hopefully the problem will not arise again.
About DMOS....I found this ... https://www.sigmaaldrich.com/content/dam/sigma-aldrich/docs/Sigma/Product_Information_Sheet/2/m1633pis.pdf
Viktor Y Butnev, your suggestion worked nicely and we solved the issue with the solubilization of the compound! Moreover, the experiment gave some interesting results! Thanks again! :-D
can you please share the protocol for this enzyme assay. Are you performing this for the diagnosis of gaucher's disease?
Khurram Shah, I used the protocol that was used in Osellame et al. 2013 Cell Metabolism.
My experiments were done to understand if GBA activity was altered in models for Parkinson's disease, not in Gaucher's disease. But also in those models, the assay was not meant to be used for the diagnosis.
Best wishes and good luck with your experiments!
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