A Dye Release Assay to Quantify Enzymatic Activity of Antimicrobial Proteins

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…

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