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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

In this manuscript, we established a high throughput method to quantify alkaline phosphatase activity in S. aureus biofilm culture from 96-well tissue culture plate

Abstract

Alkaline phosphatase (ALP) is a common enzyme expressed in both prokaryotic and eukaryotic cells. It catalyzes the hydrolysis of phosphate monoesters from many molecules at basic pH and plays an indispensable role in phosphate metabolism. In humans, eukaryotic ALP is one of the most frequently used enzymatic signals in diagnosing various diseases, such as cholestasis and rickets. In S. aureus, ALP is detected exclusively on the cell membrane; it is also expressed as a secretory form as well. Yet, little is known about its function in biofilm formation.

The purpose of this manuscript is to develop a quick and reliable assay to measure ALP activity in S. aureus biofilm that does not require protein isolation. Using p-nitrophenyl phosphate (pNPP) as a substrate, we measured ALP activity in S. aureus biofilm formed in 96-well tissue culture plates. Activity was based on the formation of the soluble reaction product measured by 405 nm absorbance. The high throughput nature of the 96 well tissue culture plate method provides a sensitive and reproducible method for ALP activity assays. The same experimental set up can also be extended to measure other extracellular molecular markers related to biofilm formation.

Introduction

Alkaline phosphatase (ALP) is ubiquitously expressed in both prokaryotic and eukaryotic cells1. It can catalyze the hydrolysis of monophosphate from different molecules such as nucleotides, proteins, alkaloids, phosphate esters and anhydrides of phosphoric acid. In humans, eukaryotic ALP is present in many tissues including liver, bone, intestine and placenta2. It plays important roles in protein phosphorylation, cell growth, apoptosis, stem cell processes as well as normal skeletal mineralization. Eukaryotic ALP is also a key serum indicator for the presence of diseases in bone, liver, and other tissues/organs when elev....

Protocol

1. Medium Preparation

  1. Prepare 1 L of Tryptic Soy Broth (TSB): Into 1 L of distilled water, add 15 g of pancreatic digest of casein, 5 g of papaic digest of soybean meal, 3 g of sodium chloride, 2.5 g of dextrose, and 2.5 g of dipotassium phosphate. Sterilize before use.
  2. For Tryptic Soy Agar (TSA), add 15 g of agar to 1 L of TSB, autoclave. Then let it cool down to room temperature. Next, pour at a ratio of 20 mL per Petri dish (100 mm x 15 mm).
  3. Biofilm culture medium: Add 10 g of glucose t.......

Representative Results

Figure 1 shows a representative result of ALP activity from biofilm cultures of S. aureus in 96 well tissue culture plates. 75 μL of commercially available pNPP solution was added to each well and incubated at room temperature. After incubation for different times (0 min, 15 min, 30 min, 45 min, 60 min, and 75 min, respectively) 75 μL of 5 M NaOH was added to stop the reaction. The product was then measured in a 96 well plate reader at 405 .......

Discussion

In our assay, we used pNPP as the ALP substrate. This is a working solution designed for ELISA and no dilution is needed. After hydrolysis by ALP, a yellow product develops and can be measured at 405 nm. At the end of the enzymatic reaction, we briefly centrifuged the 96 well plate and transferred the supernatant to a fresh 96 well plate to measure absorbance using the plate reader. We found that this extra centrifugation step is critical, since it increases the consistency of absorbance at 405 nm, probably by eliminatin.......

Acknowledgements

We thank William Rainey Harper College and the University of Illinois at Chicago for the facility to conduct these experiments. We also thank McGraw Hill Foundation for their generous support.

....

Materials

NameCompanyCatalog NumberComments
AgarVWR9002-18-0
Eppendorf CentrifugeThomas Scientific5810
GluoseVWR50-99-7
NaOH pelletsVWRSS0550-500GR
Para-nitrophenylphosphate (pNPP) SigmaP7998-100MLTypical concentrations of pNPP liquid substrates, often used in enzyme-linked immunesorbent assays (ELISA), range between 10 to 50 mM. Similar to most ready-to-use pNPP liquid substrates like the one used here, the exact pNPP concentration is not disclosed due to its proprietary nature.
10X PBS, pH7.4.
173 mM NaCl,
2.7 mM KCl,
8 mM Na2HPO4,
2 mM KH2PO4
SigmaP3288-1VL
Plate ReaderBiotekELx808
S. aureusATCCATCC25923
Tryptic Soy Agar       15g / L TSBVWR9002-18-0
Tryptic Soy Broth:      g/L
Pancreatic Digest of Casein............ 15.0
Papaic Digest of Soyben Meal.........5.00
Sodium chloride.............................. 3.00
Dextrose........................................  2.50
Dipotassium phosphate..................2.50
VWR90006-098
96 well tissue culture platesBD6902D09U shaped bottom

References

  1. Coleman, J. Structure and mechanism of alkaline phosphatase. Annual Review of Biophysics and Biomolecular Structure. 21, 441-483 (1992).
  2. Sharma, U., Pal, D., Prasad, R. Alkaline Phosphatase: An Overview. Indian Journal of Clinical Bioch....

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Alkaline Phosphatase ActivityS AureusBiofilmColorimetric AnalysisHigh throughputTSBTSAGlucose96 well PlatePNPPSodium Hydroxide

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