Reverse Zymmogram Analysis for the Detection of Protease Inhibitor Activity

Materials and Reagents

1. Protein extract or purified protein (1-5 μg)
   
2. Aprotinin (Sigma-Aldrich, catalog number: A-3886 )
   
3. Subtilisin (Sigma-Aldrich, catalog number: P-5380 )
   
4. 30% Acrylamide stock (29:1 acrylamide:bisacrylamide) (Bio-Rad Laboratories)
   
5. TEMED (Sigma-Aldrich)
   
6. Ammonium persulfate (Bio-Rad Laboratories)
   
7. SDS (Bio-Rad Laboratories)
   
8. Tris base (Sigma-Aldrich)
   
9. Gelatin (Sigma-Aldrich)
   
10. Bromophenol Blue (Sigma-Aldrich)
    
11. β-mercaptoethanol (Sigma-Aldrich)
    
12. Glycine (Sigma-Aldrich)
    
13. EDTA (JT Baker)
    
14. Glycerol (JT Baker)
    
15. Pre-stain Protein Standard (Bio-Rad Laboratories)
    
16. Coomassie blue G250 (Sigma-Aldrich)
    
17. Ethanol (JT Baker)
    
18. Phosphoric acid (Sigma-Aldrich)
    
19. Triton X-100 (Sigma-Aldrich)
    
20. Separating gel buffer (8x) (see Recipes)
    
21. Stacking gel buffer (4x) (see Recipes)
    
22. Gelatin-SDS-PAGE Separating gel (see Recipes)
    
23. Stacking gel (see Recipes)
    
24. Sample loading buffer (see Recipes)
    
25. Laemmli Reservoir buffer (see Recipes)
    
26. Stain solution (4 L) (see Recipes)
    

Equipment

1. Protein mini gel cassettes (Bio-Rad Laboratories)
   
2. Power supply
   
3. Orbital shaker
   
4. Incubator
   

Procedure

1. Preparation of the gelatin-SDS-PAGE gel
   1. Clean and completely dry glass plates, combs, spacers (0.75-1 mm), and assemble the gel cassette.
      
   2. Dissolve 1% gelatin in dH₂O by heating, and keep warm to avoid gelling.
      
   3. Prepare the 12.5% separating gel, replacing the corresponding volume of water with the gelatin solution to a final concentration of 0.1%. Mix well and quickly transfer to the casting chamber to avoid the uneven gelling of the gelatin. Add a small layer of water or isopropanol prior to polymerization to level the gel.
      
   4. Once the gel has polymerized remove the water or isopropanol layer and dry off as much as possible by using a filter paper. Prepare and pour the stacking solution into the casting chamber and insert the comb.
      
   5. Sample Preparation.
      
   6. Add the same volume of 2x protein sample loading buffer to each protein extract to be tested, and mix. Do not heat at any time.
      
   7. Use a protein-based protease inhibitor as a positive control for the reaction, such as: aprotinin for subtilisin, or trypsin inhibitor for trypsin and load it at a similar concentration to the protein samples tested.
      
2. Electrophoresis. Performed at 100 V for approximately 2.5 h at 4 °C.
3. Eliminating SDS from the gel. SDS in the gel may interfere with activity of the protease inhibitor to be tested, so it must be removed from gel before treating the gel with the protease. Removal is carried out by:
   1. Rinsing twice with 30 ml of 2.5% (v/v) Triton X-100 solution, for 10 min each with agitation.
      
   2. Rinsing twice with 30 ml of 2.5% (v/v) Triton X-100 + 50 mM Tris-HCl (pH 7.4) solution, for 10 min each with agitation.
      
   3. Rinsing with 30 ml of 50 mM Tris-HCl (pH 7.4), for 10 min with agitation.
      
4. For protein digestion, incubate the gel for 2 h at 37 °C, in a buffer solution containing 1.4 U of subtilisin in 50 ml of 50 mM Tris-HCl (pH 7.4), 200 mM NaCl.
5. For fixation, place the gel in a 10% methanol, 10% acetic acid solution with gentle shaking for 30 min. Discard the solution.
   
6. Detection of proteolysis inhibitors. Stain the gel with Coomassie Brilliant Blue, by adding approximately 100 ml of stain solution and leaving it in an orbital shaker overnight. Discard the stain and rinse the gel with water until the background gel becomes clear, which indicates the efficient degradation of the copolymerized gelatin. The presence of stained bands indicates areas where gelatin was protected from degradation by the activity of a protease inhibitor (hence “protection bands”).
   
7. Commonly a twin gel, lacking gelatin is run in parallel as a control.
   
8. If an antibody for the tested protease inhibitor is available, the digested gel can be transferred and an immunoblot assay can be performed.

Recipes

1. Separating gel buffer (8x)
   3 M Tris-HCl (pH 8.8)
   
2. Stacking gel buffer (4x)
   0.5 M Tris-HCl (pH 6.8)
   
3. Gelatin-SDS-PAGE Separating gel
   Add the following solutions (total volume: 5 ml)
   

   30% acrylamide/bisacrylamide	2.08 ml
   dH2O	1.72 ml
   Separating gel buffer (8x)	0.625 ml
   20% SDS	25 μl
   1% gelatin solution	0.5 ml
   10% ammonium persulfate	25 μl
   TEMED (add it right before pouring the gel)	5 μl

4. Stacking gel (total volume: 2.5 ml)
   

   30% acrylamide/bisacrylamide	0.5 ml
   dH2O	1.375 ml
   Stacking gel buffer (4x)	0.625 ml
   20% (w/v) SDS	15 μl
   10% ammonium persulfate	15 μl
   TEMED	5 μl

5. Sample loading buffer
   0.12 M Tris
   10% 2-mercaptoethanol
   20% (v/v) glycerol
   2 mg/ml Bromphenol blue
   
6. Laemmli Reservoir buffer
   25 mM Tris base
   0.192 M Glycine
   0.1% (w/v) SDS
   pH around 8.3
   Should not require adjustment
   Store at room temperature
   
7. Stain solution (4 L)
   

   Coomassie blue R250	3.2 g
   Ethanol	800 ml
   Phosphoric acid	64 ml
   Ammonium sulphate	320 g

   Dissolve Coomassie blue G250 in ethanol and add phosphoric acid. Dissolve the ammonium sulphate in water and add to the mix. Adjust final volume with water.
   

Acknowledgments

This protocol is adapted from Jimenez-Duran et al. (2013).

References

1. Jimenez-Duran, K., McClure, B., Garcia-Campusano, F., Rodriguez-Sotres, R., Cisneros, J., Busot, G. and Cruz-Garcia, F. (2013). NaStEP: a proteinase inhibitor essential to self-incompatibility and a positive regulator of HT-B stability in Nicotiana alata pollen tubes. Plant Physiol 161(1): 97-107.
   
2. Laemmli, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227(5259): 680-685.
   
3. Lantz, M. S. and Ciborowski, P. (1994). Zymographic techniques for detection and characterization of microbial proteases. Methods Enzymol 235: 563-594.