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…
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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 |
