Hi!
My team used CaCl2 assay plate to test the activity of lipase. Basically, the recipe that we have used is as described: LB agar base, 2 % Tween 20 and 5 mM CaCl2. The sample that we used are crude protein extract and purified protein fraction. Problem is we get a variation of result. Sometimes, on the plate, post incubation, we can see a halo clear zone developed first. During other time, we can see Ca precipitation in the agar. On the other time we can see both the clear zone and protein precipitation as well. The examples are shown in the figures attached. Why does that happened?
Thank you!
Fatin Safiudin The variation observed in your CaCl₂-Tween 20 lipase assay plates—ranging from clear halos, calcium precipitation, or both is due to differences in enzyme concentration, substrate hydrolysis rate, and interactions between fatty acids and calcium ions. When lipase hydrolyzes Tween 20, it releases fatty acids that can react with Ca²⁺ to form insoluble calcium soaps, seen as opaque white precipitates; however, at higher enzyme activity or with better diffusion, the hydrolysis may proceed extensively, preventing or dissolving the precipitate and forming a clear halo instead. Crude extracts may contain interfering components like salts, proteases, or detergents that influence local pH or protein stability, while purified proteins may precipitate directly or alter diffusion patterns. These outcomes are further affected by agar composition, moisture, and minor inconsistencies in plate preparation or incubation, all contributing to the observed variability.
Thank you Rajarethinam Kumar for the answer :)) Can you elaborate more on how at higher enzyme activity the Ca soap formed dissolved and/or prevented from forming leading to clear halo exclusively instead.
Thank you in advance
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