Reverse Zymmogram Analysis for the Detection of Protease Inhibitor Activity

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Plant Physiology
Jan 2013



This protocol describes a gel-based procedure to detect protease inhibitor activity. In this method gelatin is used as a substrate for proteolysis and is copolymerized within the polyacrylamide matrix. Protein extracts are fractioned by SDS-PAGE and then the gel is treated with the protease of interest, which degrades gelatin, except in the areas where inhibitory activity is present. Inhibition of protease activity appears as colored bands against a clear background after staining with Coomassie Brilliant Blue (Figure 1). The effectiveness of the assay is dependent on the capacity of the protease inhibitor to refold after SDS-PAGE fractionation. Alternatively, it can be performed using native (PAGE) gels. Although the protocol presented here has been standardized to test for subtilisin inhibitory activity, it can easily be adapted to test other proteases and protease inhibitors.

Figure 1. Protease inhibitor activity by zymogram analysis of different purified fractions and a protein crude extract. Zymogram was performed after SDS-PAGE. One microgram of protein was loaded to each lane. CE: Crude extract; QS: Fraction from anion exchange chromatography (Q-sepharose); Fractions 35-36: Obtained after a size exclusion chromatography (Superdex 200). M: Molecular markers. Arrows indicate inhibition 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)


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


  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.


  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
    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
    1.375 ml
    Stacking gel buffer (4x)
    0.625 ml
    20% (w/v) SDS
    15 μl
    10% ammonium persulfate
    15 μl
    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
    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.


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


  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.


该协议描述了基于凝胶的程序来检测蛋白酶抑制剂活性。 在该方法中,明胶用作蛋白水解的底物并在聚丙烯酰胺基质内共聚。 通过SDS-PAGE分级分离蛋白质提取物,然后用目标蛋白酶处理凝胶,除了存在抑制活性的区域外,其降解明胶。 在用考马斯亮蓝染色后,蛋白酶活性的抑制表现为针对透明背景的有色条带(图1)。 测定的有效性取决于蛋白酶抑制剂在SDS-PAGE分离后再折叠的能力。 或者,其可以使用天然(PAGE)凝胶进行。 虽然这里提出的协议已经标准化以测试枯草杆菌蛋白酶抑制活性,它可以很容易地适应测试其他蛋白酶和蛋白酶抑制剂。

图1.不同纯化级分和蛋白质粗提物的酶谱分析的蛋白酶抑制剂活性。在SDS-PAGE后进行酶谱。 将1微克蛋白质加载到每个泳道。 CE:粗提取物; QS:来自阴离子交换层析(Q-sepharose)的部分; 级分35-36:在尺寸排阻色谱(Superdex 200)后获得。 M:分子标记。 箭头表示抑制活性。


  1. 蛋白质提取物或纯化蛋白(1-5μg)
  2. 抑肽酶(Sigma-Aldrich,目录号:A-3886)
  3. 枯草杆菌蛋白酶(Sigma-Aldrich,目录号:P-5380)
  4. 30%丙烯酰胺原液(29:1丙烯酰胺:双丙烯酰胺)(Bio-Rad Laboratories)
  5. TEMED(Sigma-Aldrich)
  6. 过硫酸铵(Bio-Rad Laboratories)
  7. SDS(Bio-Rad Laboratories)
  8. Tris碱(Sigma-Aldrich)
  9. 明胶(Sigma-Aldrich)
  10. 溴酚蓝(Sigma-Aldrich)
  11. β-巯基乙醇(Sigma-Aldrich)
  12. 甘氨酸(Sigma-Aldrich)
  13. EDTA(JT Baker)
  14. 甘油(JT Baker)
  15. 预先染色蛋白标准品(Bio-Rad Laboratories)
  16. 考马斯蓝G250(Sigma-Aldrich)
  17. 乙醇(JT Baker)
  18. 磷酸(Sigma-Aldrich)
  19. Triton X-100(Sigma-Aldrich)
  20. 分离凝胶缓冲液(8x)(参见配方)
  21. 堆叠胶缓冲液(4x)(参见配方)
  22. 明胶SDS-PAGE分离凝胶(参见配方)
  23. 堆叠胶(见配方)
  24. 样品加载缓冲液(参见配方)
  25. Laemmli水库缓冲区(参见配方)
  26. 染色溶液(4 L)(参见配方)


  1. 蛋白质微型凝胶盒(Bio-Rad Laboratories)
  2. 电源
  3. 轨道振动器
  4. 孵化器


  1. 明胶SDS-PAGE凝胶的制备
    1. 清洁并完全干燥玻璃板,梳子,垫片(0.75-1 mm),然后组装凝胶盒
    2. 通过加热将1%明胶溶于dH 2 O中,并保温以避免胶凝。
    3. 制备12.5%分离凝胶,用明胶溶液代替相应体积的水至终浓度为0.1%。 混合好并快速转移到流延室,以避免明胶的不均匀胶凝。 在聚合之前加入小层水或异丙醇以使凝胶变平。
    4. 一旦凝胶聚合,除去水或异丙醇层并通过使用滤纸尽可能干燥。 准备并将堆叠溶液倒入浇铸室并插入梳子
    5. 样品制备。
    6. 向待测试的每种蛋白质提取物中加入相同体积的2x蛋白质样品上样缓冲液,并混合。 不要随时加热。
    7. 使用基于蛋白质的蛋白酶抑制剂作为反应的阳性对照,例如:枯草杆菌蛋白酶的抑肽酶或胰蛋白酶的胰蛋白酶抑制剂,并以与测试的蛋白质样品相似的浓度加载它。
  2. 电泳。 在4℃下在100V下进行约2.5小时。
  3. 从凝胶中除去SDS。 凝胶中的SDS可能干扰待测试的蛋白酶抑制剂的活性,因此在用蛋白酶处理凝胶之前必须从凝胶中除去。 去除通过以下步骤进行:
    1. 用30ml 2.5%(v/v)Triton X-100溶液冲洗两次,每次搅拌10分钟。
    2. 用30ml 2.5%(v/v)Triton X-100 + 50mM Tris-HCl(pH7.4)溶液冲洗两次,每次搅拌10分钟。
    3. 用30ml 50mM Tris-HCl(pH 7.4)冲洗10分钟,同时搅拌。
  4. 对于蛋白质消化,在37℃下,在含有1.4U枯草杆菌蛋白酶在50ml 50mM Tris-HCl(pH 7.4),200mM NaCl的缓冲溶液中孵育凝胶2小时。
  5. 对于固定,将凝胶置于10%甲醇,10%乙酸溶液中,轻轻摇动30分钟。舍弃解决方案。
  6. 蛋白水解抑制剂的检测。用考马斯亮蓝染色凝胶,通过加入约100ml染色溶液,并将其留在轨道振荡器中过夜。弃去污渍,用水冲洗凝胶,直到背景凝胶变得澄清,这表明共聚明胶的有效降解。染色条带的存在表明通过蛋白酶抑制剂的活性(因此"保护条带")保护明胶免于降解的区域。
  7. 通常,缺少明胶的双凝胶作为对照平行进行
  8. 如果用于测试的蛋白酶抑制剂的抗体可用,则可以转移消化的凝胶并进行免疫印迹测定。


  1. 分离凝胶缓冲液(8x)
    3 M Tris-HCl(pH 8.8)
  2. 堆叠凝胶缓冲液(4x)
    0.5M Tris-HCl(pH 6.8)
  3. 明胶SDS-PAGE分离凝胶
    30%丙烯酰胺/双丙烯酰胺 2.08 ml
    dH 2 2 O 1.72 ml
    0.625 ml
    1%明胶溶液 0.5 ml
  4. 堆积凝胶(总体积:2.5ml)
    30%丙烯酰胺/双丙烯酰胺 0.5 ml
    dH 2 2 O 1.375 ml
    0.625 ml
  5. 样品加载缓冲区
    0.12 M Tris
    10%2-巯基乙醇 20%(v/v)甘油 2mg/ml溴酚蓝
  6. Laemmli水库缓冲区
    25 mM Tris碱
    0.192 M甘氨酸
  7. 染色溶液(4L)
    800 ml
    64 ml
    将考马斯蓝G250溶解在乙醇中,加入磷酸。 将硫酸铵溶解在水中并加入到混合物中。 用水调节最终体积。




  1. Jimenez-Duran,K.,McClure,B.,Garcia-Campusano,F.,Rodriguez-Sotres,R.,Cisneros,J.,Busot,G.and Cruz-Garcia, NaStEP:一种对自身不相容性至关重要的蛋白酶抑制剂和HT-B稳定性的正调节剂 em> Nicotiana alata 花粉管。植物生理学 161(1):97-107。
  2. Laemmli,U.K。(1970)。 在噬菌体T4头部装配过程中切割结构蛋白。 自然 227(5259):680-685
  3. Lantz,M.S.and Ciborowski,P。(1994)。 用于检测和表征微生物蛋白酶的酶谱技术方法Enzymol em> 235:563-594。
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Copyright: © 2013 The Authors; exclusive licensee Bio-protocol LLC.
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Bernal, L., García-Campusano, F., Nájera, E. and Cruz-García, F. (2013). Reverse Zymmogram Analysis for the Detection of Protease Inhibitor Activity. Bio-protocol 3(16): e856. DOI: 10.21769/BioProtoc.856.
  2. 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.