Biochemistry

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Authors: Naxin Jiang, Nguan Soon Tan, Bow Ho & Jeak Ling Ding 

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.

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.

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.

Associated Publications

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

Author information

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.

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