My study is on lipase and I planned to used p-NP palmitate method as the activity assay. The recipe includes Tris HCl buffer pH 9 + Triton X-100 + isopropanol. I stirred the mixture with heat then the color gradually turns greenish yellowish. Color change to yellowish hue indicate positive lipase activity which means that the mixture might be contaminated but I am not sure how and when.
FYI, I used a newly opened p-NP palmitate and freshly made Tris buffer. Should I sterilize everything before hand?
It us unlikely that your substrate became contaminated with lipase accidentally if you were using clean apparatus and containers. Most likely, the problem is the alkaline pH combined with heat causing spontaneous hydrolysis, as suggested by Nick Gee
Reduce the initial pH of the enzymatic assay and retest...Observe the color-forming variation at pH 7-8-9...These must be amenable to lipase activity. The high alkaline conditions may occur as non-specific ester bond hydrolysis, also induced by heat. Consider the alkaline hydrolysis of the fragile ester bond between the pnp and conjugated palmitate...
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