Methods in Enzymology Vol.57 Bioluminescence and Chemiluminescence

This chapter presents a procedure for purification of the light-emitting system that consists of gel filtration for removal of compounds of low-molecular weight, followed by purification of luciferase by ion-exchange chromatography. The procedure is preferable for routine preparations because a reasonably good purification is obtained in minimal time and under uncritical conditions. The purification of extracts of firefly tails for use in the analysis of adenosine triphosphate (ATP) requires methods that can be applied to rather small amounts of material. The fractionation requirements are, however, well defined: (1) removal of ATP and other substances of low-molecular weight to lower the blank values, and (2) removal of interfering enzymes to decrease the systematic errors of the analysis. The substances of low-molecular weight are easily removed by column-gel filtration on low-porosity Sephadex gels. Luciferin and dehydroluciferin are strongly adsorbed on this material from neutral buffers. The ATP analysis has numerous systematic errors.

• Series: Methods in Enzymology (Book 57)
• Hardcover: 653 pages
• Publisher: Academic Press;
  
• Language: English
• ISBN-10: 0121819574
• ISBN-13: 978-0121819576
• Product Dimensions: 6 x 1.4 x 9 inches

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