Azocoll protease activity assay
Lab/Group: Jeak Ling DING’s lab (National University of Singapore)
Related Journal & Article Information
Journal: Nature Immunology
Introduction
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.
Materials
Reagents
Equipment
Procedure
1. Resuspend 75 mg of Azocoll with 50 ml of 0.01 M PBS, pH 7.4.
2. Gently swirl the suspension at room temperature for 2 h.
3. Centrifuge the mixture at 10,000 x g at room temperature for 10 min.
4. Remove the supernatant containing the degraded peptide which may cause high background in protease activity assay.
5. Repeat steps 1 to 4.
6. Resuspend the washed Azocoll reagent in 50 ml of 0.01 M PBS, pH 7.4. This is ready for further protease activity assay.
7. 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.
8. Centrifuge the reaction mixture at 10,000 x g at room temperature for 10 min.
9. Transfer the supernatant to a 96 well microtitre plate.
10. Monitor the absorbance at 550 nm using ELISA reader.
11. The protease activity is presented as the A550 of the incubation supernatant.
Troubleshooting
Critical Steps
Anticipated Results
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.
References
Acknowledgements
Keywords
protease; azocoll