A Dye Release Assay to Quantify Enzymatic Activity of Antimicrobial Proteins

Published: February 29, 2024

Abstract

Source: Farris, M. H. et al., Qualitative and Quantitative Assays for Detection and Characterization of Protein Antimicrobials. J. Vis. Exp. (2016)

This video demonstrates the dye release assay for quantifying the enzymatic activity of antimicrobial proteins. The antimicrobial protein degrades the bacteria's dye-labeled cell wall and releases the dye molecules turning the solution blue. The absorbance of the solution correlates with the enzymatic activity of the antimicrobial protein.

Protocol

1. Preparation of Bacteria NOTE: Whole-cell bacterial substrates and purified peptidoglycans are used as enzyme substrates in both the microslide diffusion assay and the dye-release assay. These substrates require preparation prior to conducting the enzyme reactions. The following protocol describes their preparation. Whole Bacterial Cell Substrate Produce bacterial cell substrate by inoculating 500 ml of nutrient broth with a 2 ml over…

開示

The authors have nothing to disclose.

Materials

48-well flat-bottom microplate with low evaporation lid Becton Dickinson 3078
96-well flat-bottom microplate (Costar 3595) Corning, Inc. 3595
agarose Fisher Scientific BP162-100
Bacillus subtilis 168 American Type Culture Collection 23857
C1000 Touch Thermal Cycler Bio-Rad
McFarland equivalence standard (2.0) Fisher Scientific R20412
Nutrient broth Fisher Scientific S25959B
Phosphate-buffered saline (1×) Teknova P0200
Pierce BCA Protein Assay Kit Life Technologies 23227
Synergy HT microplate spectrophotometer BioTek Instruments, Inc.
Remazol brilliant blue R dye (RBB) Sigma-Aldrich R8001
Sodium hydroxide (NaOH) Fisher Scientific S318-500
UltraPure distilled water Invitrogen 10977-015
Solution
Nutrient broth medium
4 g nutrient broth
500 ml Type I water
250mM sodium hydroxide (NaOH) solution
1 g NaOH
99 ml Type I water
200mM Remazol brilliant blue R dye (RBB)
1.25 g RBB
98.75 ml 250 mM NaOH solution

タグ

Play Video

記事を引用
A Dye Release Assay to Quantify Enzymatic Activity of Antimicrobial Proteins. J. Vis. Exp. (Pending Publication), e21975, doi: (2024).

View Video