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.
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.
2. Substrate Labeling – Remazol Brilliant Blue R Dye Labeling
NOTE: In the dye-release assay, the substrate is covalently linked to Remazol brilliant blue R dye. The following protocol describes the preparation of dyed enzyme substrates.
3. Quantitative Dye-release Assay
NOTE: During the dye-release assay, the hydrolysis of RBB-dyed substrate in the enzymatic reaction releases dyed products into the reaction supernatant. Colorimetric measurement of the amount of dye released indicates the amount of enzymatic activity present within the sample for a given enzyme. The quantitative method allows the comparison of different enzymes across substrates and allows for variation of environmental conditions influencing the enzymatic reaction (e.g., temperature, salinity, and pH). The following protocol describes the preparation and performance of the dye-release assay for quantitatively detecting the enzymatic activity of protein antimicrobials.
The authors have nothing to disclose.
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 |