Has anyone experienced difficulties during laccase assay measurements?
I have an enzymatic extract from a solid-state fermentation of sugarcane bagasse. The extract is dark, which interferes with the use of ABTS as a substrate for measuring laccase activity.
The ABTS is taking an unusually long time to be oxidized (more than 24 hours). I have already tried dialysis, but it still hasn't turned green or blue.
Does anyone have any recommendations?
11/14/24
Dear Clara,
Is it possible for you to concentrate the enzyme by precipitating if from the extract? If there is enough protein in the extract, you may be able to precipitate the enzyme (as well as other proteins) using ammonium sulfate ((NH4)2SO4). Ammonium sulfate (AS) at ~65-70% is an effective concentration for precipitating proteins. It is an old technique, but it does work. The AS competes w/ proteins (and other solutes) for the available water, causing proteins to precipitate. This can be recovered by centrifugation. Much of the unwanted material (hopefully the dark material in your extract) will remain in solution. If this works, you can recover the protein precipitate and dissolve it in an appropriate buffer, then assay it for activity.
I hope this information helps you.
Bill Colonna, Dept. Food Science & Human Nutrition, Iowa State University, Ames, Iowa, USA [email protected]
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