Bioluminescence assay

BY, JAGRUTI RAVINDRA SUTAR

1. BIOLUMINESCENCE ASSAY Presented by: Jagruti Ravindra Sutar. M. Pharm. 1 st year. Sem I. Pharmaceutics Roll No- 13 Subject- Modern Pharmceutical Analytical Technique

2. CONTENT Introduction Principle Procedure Instrumentation Application

3. INTRODUCTION Bioluminescence is the production and emission of light by a living organism. It is a form of chemiluminescence. Bioluminescence occurs widely in marine vertebrates and invertebrates, as well as in some fungi, microorganisms including some bioluminescent bacteria and terrestrial invertebrates such as fireflies. Bioluminescence assays involve the use of the property of bioluminescence for measuring cell proliferation, apoptosis, drug metabolism, kinas activity, etc.

4. PRINCIPLE Bioluminescence is a form of chemiluminescence where light energy is released by a chemical reaction. This reaction involves a light-emitting pigment, the luciferin , and a luciferase, the enzyme component. Luciferin + Oxygen Oxyluciferin + Light energy ( AMP, PP and Carbon dioxide ) Mg++ ATP

5. PROCEDURE Aspirate medium of cells Rinse with PBS Add sufficient amount of Glo-lysis buffer Rock the plate slowly to complete coverage of the cells Incubate for 5 min to allow cell lysis

6. Transfer the lysate to new tube Centrifuge for 10 min at 1000g Place 50 µg Bright glow assay reagent into luminometer plate Add same volume of lysate Measure luminescence with Luminometer

7. INSTRUMENTATION Fig: Luminometer Microplate Monochromater & filters Detector

8. Fig: Microplate

9. APPLICATION Food testing using ATP technology Bioluminescent imaging Development of new treatment for antibiotic resistant tuberculosis Discovery of green protein