Materials and Reagents

Protein extract or purified protein (1-5 μg)
Aprotinin (Sigma-Aldrich, catalog number: A-3886 )
Subtilisin (Sigma-Aldrich, catalog number: P-5380 )
30% Acrylamide stock (29:1 acrylamide:bisacrylamide) (Bio-Rad Laboratories)
TEMED (Sigma-Aldrich)
Ammonium persulfate (Bio-Rad Laboratories)
SDS (Bio-Rad Laboratories)
Tris base (Sigma-Aldrich)
Gelatin (Sigma-Aldrich)
Bromophenol Blue (Sigma-Aldrich)
β-mercaptoethanol (Sigma-Aldrich)
Glycine (Sigma-Aldrich)
EDTA (JT Baker)
Glycerol (JT Baker)
Pre-stain Protein Standard (Bio-Rad Laboratories)
Coomassie blue G250 (Sigma-Aldrich)
Ethanol (JT Baker)
Phosphoric acid (Sigma-Aldrich)
Triton X-100 (Sigma-Aldrich)
Separating gel buffer (8x) (see Recipes)
Stacking gel buffer (4x) (see Recipes)
Gelatin-SDS-PAGE Separating gel (see Recipes)
Stacking gel (see Recipes)
Sample loading buffer (see Recipes)
Laemmli Reservoir buffer (see Recipes)
Stain solution (4 L) (see Recipes)

Equipment

Protein mini gel cassettes (Bio-Rad Laboratories)
Power supply
Orbital shaker
Incubator

Procedure

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.
Electrophoresis. Performed at 100 V for approximately 2.5 h at 4 °C.
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.
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.
For fixation, place the gel in a 10% methanol, 10% acetic acid solution with gentle shaking for 30 min. Discard the solution.
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”).
Commonly a twin gel, lacking gelatin is run in parallel as a control.
If an antibody for the tested protease inhibitor is available, the digested gel can be transferred and an immunoblot assay can be performed.

Recipes

Separating gel buffer (8x)
3 M Tris-HCl (pH 8.8)
Stacking gel buffer (4x)
0.5 M Tris-HCl (pH 6.8)
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
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
Sample loading buffer
0.12 M Tris
10% 2-mercaptoethanol
20% (v/v) glycerol
2 mg/ml Bromphenol blue
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
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

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.
Laemmli, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227(5259): 680-685.
Lantz, M. S. and Ciborowski, P. (1994). Zymographic techniques for detection and characterization of microbial proteases. Methods Enzymol 235: 563-594.