PPi Release Assay Works Well

September 08, 2014

Overall

Quality of Results

Ease-of-Optimization

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Laboratory Services Division, University of Guelph
MMD
Senior Technician

Company:

Lonza

Product Name:

PPiLight™ Inorganic Pyrophosphate Assay

Catalog Number:

LT07-610

I used the Lonza PPi release assay to measure the activity of a pyrophosphorylase. I also compared the activity to a suspect pyrophosphorylase, however it did not have any activity and worked well as a negative control. The assay was predominantly run with purified protein, however co-purified protein fractions from a size-exclusion column also showed activity. The repeatability was good, and I didn’t have any major problems. I received the product through the Canadian distributor Cedar Lane.

Experimental Design and Results Summary

Application

Enzyme Kinetics

Starting Material

Purified protein, co-factor reagents

Protocol Overview

The PPiLight™ Assay is a non-radioactive bioluminescent assay for the detection of inorganic pyrophosphate. In the presence of PPi the detection reagent catalyses the conversion of AMP to ATP. The assay uses luciferase, which produces light from the newly formed ATP and luciferin.

Tips

Keep all reagents on ice, and thaw reagents on ice as directed, do not heat detection reagents to thaw.

Results Summary

I used the assay to describe the activity of a pyrophosphorylase, the regression statistics were always over 0.85 when using different concentrations of a substrate sugar.

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Summary

The Good

Fairly easy setup, good repeatability, pretty cheap, luminescence is easy to read

The Bad

Need to thaw detection reagents, and they have to be mixed fresh each time

The Bottom Line

The product is easy to use, the results are reproducible, the reaction is fairly rugged. I ran into no problems with the kit.

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