Authors: Naxin Jiang, Nguan Soon Tan, Bow Ho & Jeak Ling Ding
Azocoll, the Azo dye-impregnated collagen (Sigma) is used as a chromogenic non-specific substrate for protease activity assay. Upon proteolysis, soluble peptide fragments which is purple in color due to Azo dye-impregnation, are released and can be detected by absorbance at 550 nm.
- Resuspend 75 mg of Azocoll with 50 ml of 0.01 M PBS, pH 7.4.
- Gently swirl the suspension at room temperature for 2 h.
- Centrifuge the mixture at 10,000 x g at room temperature for 10 min.
- Remove the supernatant containing the degraded peptide which may cause high background in protease activity assay.
- Repeat steps 1 to 4.
- Resuspend the washed Azocoll reagent in 50 ml of 0.01 M PBS, pH 7.4. This is ready for further protease activity assay.
- For protease activity assay, incubate 100 μl of the protease under examination with 400 μl of the Azocoll reagents from step 6, under gentle end-to-end rotation at room temperature for 2 h. Set up triplicates for each sample.
- Centrifuge the reaction mixture at 10,000 x g at room temperature for 10 min.
- Transfer the supernatant to a 96 well microtitre plate.
- Monitor the absorbance at 550 nm using ELISA reader.
- The protease activity is presented as the A550 of the incubation supernatant.
For Pseudomonas aeruginosa, the typical readout of the extracellular protease activity assay is A550=0.4-0.7 for PAO-Iglewski, and A550<0.05 for PAO-B1A1.
Respiratory protein–generated reactive oxygen species as an antimicrobial strategy, Naxin Jiang, Nguan Soon Tan, Bow Ho, and Jeak Ling Ding, Nature Immunology 8 (10) 1114 - 1122 26/08/2007 doi:10.1038/ni1501
Naxin Jiang , Nguan Soon Tan , Bow Ho & Jeak Ling Ding, National University of Singapore
Source: Protocol Exchange (2007) doi:10.1038/nprot.2007.484. Originally published online 31 October 2007.