444
I. Moos
Electrophoresis 1991, /2, 444-445
ceipt of a predoctoral and postdoctoral jellowship from the Belgian "Instituut ter Aanmoediging van het Wetenschappelijk Onderzoek in de Nijverheid en de Landbouw': respectively. K E Broekaert is Senior Research Assistant of the National Fund f o r Scientijic Research (Belgium). Received December 12, 1990; in revised form April 3, 1991
References [I] Heeb, M. J. and Gabriel, O., Methods Enzymol. 1984, 104, 416-439. [2] Hultmark, D., Steiner, H., Rasmuson, T. and Boman, H. G., Eur. J. Biochem. 1980,106,7-16. 131 Bailey, J. A , , and Burden, R. S . , Physiol. Plant. Pathoi. 1973, 3, 171177.
Jiri Moos Institute of Molecular Genetics, Czechoslovak Academy of Sciences, Prague
[4] Grenier, J.andAsselin,A.,Mol. Plant-Microbelnteract. 1990,3,401407. [ 5 ] Hernandez-Lucas, C., Fernandez de Caleya, R. and Carbonero, P., Appl. Microbial. 1974, 28, 165-168. [6] Broekaert,W. F.,Van Parijs, J., Leyns,F., Joos, H. and Peumans,W. J., Science 1989,245, 1100-1102. [7] Broekaert, W. F.,Van Parijs, J . , Allen, A. K. and Peumans, W. J., Physiol. Mol. Plant Pathol. 1988, 33,319-331. [8] Redman, D. G. and Fisher, N., J. Sci. Fd. Agric. 1969, 20,427-432. [9] Broekaert, W. F., Terras, F. R. G., Cammue, B. P. A. and Vanderleyden, J., FEMS Microbiol. Lett. 1990, 69, 55-60. [lo] Reisfeld, R. A., Lewis, U. J. and Williams, D. E., Narure 1962, 195, 281-283. [ I l l Kovarik, A. Hlubinova, K. A., Vrbenska, A. and Prachar, J., J. Folia Biologia (Praha) 1987, 33, 253-257. [12] Bol., J . F., Lindhorst, H. J . M. and Cornelissen, B. J . C., Annu. Rev. &thopatho/. 1990,28 113-138.
Detection of gelatinolytic enzyme activities after sodium dodecyl sulfate-electrophoresis and protein blotting Visualization of proteases with gelatinolytic activity in sodium dodecyl sulfate gels is described. After conventional sodium dodecyl sulfate-polyacrylamide gel electrophoresis, proteins are transferred onto nitrocellulose membranes preincubated with 0.30'0 gelatine. During the protein electrotransfer, the proteases are renaturated and their enzymatic activity is restored. After nonspecific protein staining, bands with proteolytic activity appear as white areas on a dark background.
Usually enzymes lose their catalytic activity when submitted to electrophoresis in the presence of sodium dodecyl sulfate (SDS). However, the activity of proteases can be restored by removal of SDS with aqueous Triton X-100 [l]. Bands of proteolytic activity may then be localized by using an agar overlay containing fibrin, or plasminogen and fibrin. Heussen and Dowdle [2]and Siege1and Polakoski [3]modified this procedure using 0.1 O/o gelatine orgelatine and plasminogen copolymerized directly into SDS polyacrylamide gels. In this paper, a simple technique for the detection of proteolytic activity on nitrocellulose replicas is described. Two methods for proteolytic activity detection were used: (i) SDS-polyacrylamide gel electrophoresis (PAGE) was performed on 12% slab gel, containing 0.1 Yo copolymerized gelatine [2].After electrophoresis, gels were shaken at room temperature for 2h in 2.5% v/vTriton X-100 in water. ~ pH 8.3, Afterwards the gel was incubated in 0 . 1 glycine, adjusted with NaOH at 37 "C for 3 h,and stained in 0.1 O/o w/v Amido Black in ethanol-acetic acid-water (30: 10:60 v/v). (ii) Nitrocellulose membranes (40 pm, Serva Heidelberg, Correspondence: Dr. J. Moos, Institute of Molecular Genetics, Videnska 1083, CS-142 20 Prague 4, Czechoslovakia Abbreviations: PAGE, polyacrylamide gel electrophoresis; SDS, sodium dodecyl sulfate; TPCK, N-tosyl-L-phenylalaninechlormethylketone
0VCH Verlagsgesellschaft mbH,D-6940Weinheim, 1991
Germany) were immersed in 0.3% w/v gelatine in water and incubated for l h at room temperature.The membranes were then washed in water (2 X 5 min each) and finally immersed in 192 mM glycine, 20% v/v methanol and 25 mM Tris, pH 8.3. Proteolytic enzymes were resolved in nonreducing SDS sample buffer and subjected to SDS-PAGE [4] on 12% slab gels. SDS-PAGE was carried out on an SE 250 Mighty Small IIVertical Slab Unit (Hoefer Scientific Instruments, San Francisco, CA) using 1.5 mm thick spacers. Following electrophoresis, proteins were transferred onto gelatine-coated nitrocellulose according to the method of Towbin [5]. Transfers were performed on a TE 52 Transphor Electrophoresis Unit (Hoefer Scientific Instruments, San Francisco, CA) at 80 V for 1.5 h. The nitrocellulose replicas were then washed and blocked with 0.3% v/v Tween 20 in phosphate buffered saline (PBS) (three changes, 15 min each) and stained with a solution of colloidal gold [6]. N-Tosyl-L-phenylalanine chlormethyl ketone (TPCK) treated bovine pancreatic trypsin was purchased from Sigma (St. Louis, MO). Partially purified boar acrosin (fraction after cellulose chromatography -7) was kindly provided by Dr. Cechova. Monoclonal antibodies ACR.2 and ACR.3 [8] were used for immunodetection of boar acrosin and as a negative control, respectively. Fucose-bearing proteins were identified by biotinylated Ulex europaeus agglutinin (UEA I, Sigma) at a concentration of 10 pg/mL. Bound lectin was visualized by streptavidin-peroxidase (Amer0173-0835/91/0606-0444 $3.50+.25/0
Electrophoresis 1991, 12, 444-445
A
Detection of gelatinolytic activity
445
B Figure I . Gelatinolytic activity of trypsin after 12%SDS -PAGE. (A) Amido Black-
stained gel with copolymerized gelatine. Trypsin (1) 10 pg; (2) 1 pg; (3) 100 ng; (4) 10 ng; ( 5 ) 1 ng; (6) 100 pg; (7) 10 pg; (8) Pharmacia low molecular weight standards. (B) Colloidal gold-stained nitrocellulose replica precoated with gelatine. (1) Pharmacia low molecular weight standards: staining with Coomassie Brilliant Blue; trypsin (2) 1 ng; (3) 10 ng; (4) 100 ng; ( 5 ) 1 wg; ( 6 ) 10 clg.
sham, Buckinhamshire, UK) using the protocol recommended by the manufacturer. For total protein visualization, nitrocellulose membranes were stained with a solution of colloidal gold [6]. Molecular weights were estimated by comparing mobilities with those of low molecular weight markers (Pharmacia,Uppsala, Sweden) used in a reduced form.Al1 other electrophoretic grade chemicals were purchased from Serva.
When the procedure of Heussen and Dowdle [2] was used to determine the sensitivity of TPCK-treated trypsin, a clearly visible activity band was obtained even with only l n g of trypsin (approximately 10 Na-benzoyl-L-arginine ethyl ester (BAEE) mU,Fig. 1A, lane 5). However, the gelatine concentrated in the lower part of the gel during electrophoresis and, due to this effect, the protein markers of 14.4 and 21.5 kDa were hardlyvisible. Using the new procedure, a lower sensitivity to trypsin (10 ng) was obtained (Fig. IB, lane 3). On the other hand, protein bands without protease activity can be visualized easily. The tendency of trypsin to create trails of gelatinolysis was reduced and electrophoretic mobility of proteases was not affected by the presence o f copolymerized gelatine [2]. The main advantage of this method is the possibility to compare directly the gelatinolytic activity pattern with positions of components recognized by specific probes (Fig. 2). When partially purified boar acrosin was analyzed, the distribution o f fucose-bearing sites and positions of immunologically detected acrosin corresponded to gelatinolytic activity. However, other proteolytic activity bands were also observed (Fig. 2, lane 7). In conclusion, the new method for visualization of proteases described here is simple and time-saving and can be performed in parallel with other analytical methods for specific protein detection. Received December 14, 1990
References
Figure 2. Nitrocellulose replica of partially purified boar acrosin, scparated by SDS-PAGE undernonreducing conditions. (1) Colloidal goldstained low molecular weight standards. (2) Partially purified acrosin stained with colloidal gold. (3) Binding of biotinylated UIex europaeus agglutinin to boar acrosin. (4) Control reaction, using the streptavidin peroxidase conjugate only. ( 5 ) Binding ofmonoclonal antibodyACR.2 to acrosin. (6) Control reaction, using ACR.3 monoclonal antibody. (7) Proteolytic activity zones detected on gelatine-coated nitrocellulose.
[ I ] Granelli-Piperno, A. and Reich, F., J . Exp. Med. 1978, 148, 223-234. [2] Heussen, C. and Dowdle, E.B., Anal. Biochem. 1980, 102, 196-202. [3] Siegel, M. S . and Polakoski, K. L., B i d . Reprod. 1985, 32, 713-720. [4] Laemli, U. K., Narure 1970,227, 680-685. [5] Towbin, H., Staehelin,T. and Gordon, J., Proc. Natl. Acad. Sci. USA 1979, 76, 4350-4354. [6] Moeremans, M., Daneels, G. and Mey, J.D., Anal. Biochem. 1985, 145, 315-321. [7] Zelezna, B. and Cechova, D., Hoppe Seylers Z . Physiol. Chem. 1982, 363, 755-162. [XI Peknicova, J. and Moos, J., Andrologia 1990, 22, 427-435.
I. Moos
Electrophoresis 1991, /2, 444-445
ceipt of a predoctoral and postdoctoral jellowship from the Belgian "Instituut ter Aanmoediging van het Wetenschappelijk Onderzoek in de Nijverheid en de Landbouw': respectively. K E Broekaert is Senior Research Assistant of the National Fund f o r Scientijic Research (Belgium). Received December 12, 1990; in revised form April 3, 1991
References [I] Heeb, M. J. and Gabriel, O., Methods Enzymol. 1984, 104, 416-439. [2] Hultmark, D., Steiner, H., Rasmuson, T. and Boman, H. G., Eur. J. Biochem. 1980,106,7-16. 131 Bailey, J. A , , and Burden, R. S . , Physiol. Plant. Pathoi. 1973, 3, 171177.
Jiri Moos Institute of Molecular Genetics, Czechoslovak Academy of Sciences, Prague
[4] Grenier, J.andAsselin,A.,Mol. Plant-Microbelnteract. 1990,3,401407. [ 5 ] Hernandez-Lucas, C., Fernandez de Caleya, R. and Carbonero, P., Appl. Microbial. 1974, 28, 165-168. [6] Broekaert,W. F.,Van Parijs, J., Leyns,F., Joos, H. and Peumans,W. J., Science 1989,245, 1100-1102. [7] Broekaert, W. F.,Van Parijs, J . , Allen, A. K. and Peumans, W. J., Physiol. Mol. Plant Pathol. 1988, 33,319-331. [8] Redman, D. G. and Fisher, N., J. Sci. Fd. Agric. 1969, 20,427-432. [9] Broekaert, W. F., Terras, F. R. G., Cammue, B. P. A. and Vanderleyden, J., FEMS Microbiol. Lett. 1990, 69, 55-60. [lo] Reisfeld, R. A., Lewis, U. J. and Williams, D. E., Narure 1962, 195, 281-283. [ I l l Kovarik, A. Hlubinova, K. A., Vrbenska, A. and Prachar, J., J. Folia Biologia (Praha) 1987, 33, 253-257. [12] Bol., J . F., Lindhorst, H. J . M. and Cornelissen, B. J . C., Annu. Rev. &thopatho/. 1990,28 113-138.
Detection of gelatinolytic enzyme activities after sodium dodecyl sulfate-electrophoresis and protein blotting Visualization of proteases with gelatinolytic activity in sodium dodecyl sulfate gels is described. After conventional sodium dodecyl sulfate-polyacrylamide gel electrophoresis, proteins are transferred onto nitrocellulose membranes preincubated with 0.30'0 gelatine. During the protein electrotransfer, the proteases are renaturated and their enzymatic activity is restored. After nonspecific protein staining, bands with proteolytic activity appear as white areas on a dark background.
Usually enzymes lose their catalytic activity when submitted to electrophoresis in the presence of sodium dodecyl sulfate (SDS). However, the activity of proteases can be restored by removal of SDS with aqueous Triton X-100 [l]. Bands of proteolytic activity may then be localized by using an agar overlay containing fibrin, or plasminogen and fibrin. Heussen and Dowdle [2]and Siege1and Polakoski [3]modified this procedure using 0.1 O/o gelatine orgelatine and plasminogen copolymerized directly into SDS polyacrylamide gels. In this paper, a simple technique for the detection of proteolytic activity on nitrocellulose replicas is described. Two methods for proteolytic activity detection were used: (i) SDS-polyacrylamide gel electrophoresis (PAGE) was performed on 12% slab gel, containing 0.1 Yo copolymerized gelatine [2].After electrophoresis, gels were shaken at room temperature for 2h in 2.5% v/vTriton X-100 in water. ~ pH 8.3, Afterwards the gel was incubated in 0 . 1 glycine, adjusted with NaOH at 37 "C for 3 h,and stained in 0.1 O/o w/v Amido Black in ethanol-acetic acid-water (30: 10:60 v/v). (ii) Nitrocellulose membranes (40 pm, Serva Heidelberg, Correspondence: Dr. J. Moos, Institute of Molecular Genetics, Videnska 1083, CS-142 20 Prague 4, Czechoslovakia Abbreviations: PAGE, polyacrylamide gel electrophoresis; SDS, sodium dodecyl sulfate; TPCK, N-tosyl-L-phenylalaninechlormethylketone
0VCH Verlagsgesellschaft mbH,D-6940Weinheim, 1991
Germany) were immersed in 0.3% w/v gelatine in water and incubated for l h at room temperature.The membranes were then washed in water (2 X 5 min each) and finally immersed in 192 mM glycine, 20% v/v methanol and 25 mM Tris, pH 8.3. Proteolytic enzymes were resolved in nonreducing SDS sample buffer and subjected to SDS-PAGE [4] on 12% slab gels. SDS-PAGE was carried out on an SE 250 Mighty Small IIVertical Slab Unit (Hoefer Scientific Instruments, San Francisco, CA) using 1.5 mm thick spacers. Following electrophoresis, proteins were transferred onto gelatine-coated nitrocellulose according to the method of Towbin [5]. Transfers were performed on a TE 52 Transphor Electrophoresis Unit (Hoefer Scientific Instruments, San Francisco, CA) at 80 V for 1.5 h. The nitrocellulose replicas were then washed and blocked with 0.3% v/v Tween 20 in phosphate buffered saline (PBS) (three changes, 15 min each) and stained with a solution of colloidal gold [6]. N-Tosyl-L-phenylalanine chlormethyl ketone (TPCK) treated bovine pancreatic trypsin was purchased from Sigma (St. Louis, MO). Partially purified boar acrosin (fraction after cellulose chromatography -7) was kindly provided by Dr. Cechova. Monoclonal antibodies ACR.2 and ACR.3 [8] were used for immunodetection of boar acrosin and as a negative control, respectively. Fucose-bearing proteins were identified by biotinylated Ulex europaeus agglutinin (UEA I, Sigma) at a concentration of 10 pg/mL. Bound lectin was visualized by streptavidin-peroxidase (Amer0173-0835/91/0606-0444 $3.50+.25/0
Electrophoresis 1991, 12, 444-445
A
Detection of gelatinolytic activity
445
B Figure I . Gelatinolytic activity of trypsin after 12%SDS -PAGE. (A) Amido Black-
stained gel with copolymerized gelatine. Trypsin (1) 10 pg; (2) 1 pg; (3) 100 ng; (4) 10 ng; ( 5 ) 1 ng; (6) 100 pg; (7) 10 pg; (8) Pharmacia low molecular weight standards. (B) Colloidal gold-stained nitrocellulose replica precoated with gelatine. (1) Pharmacia low molecular weight standards: staining with Coomassie Brilliant Blue; trypsin (2) 1 ng; (3) 10 ng; (4) 100 ng; ( 5 ) 1 wg; ( 6 ) 10 clg.
sham, Buckinhamshire, UK) using the protocol recommended by the manufacturer. For total protein visualization, nitrocellulose membranes were stained with a solution of colloidal gold [6]. Molecular weights were estimated by comparing mobilities with those of low molecular weight markers (Pharmacia,Uppsala, Sweden) used in a reduced form.Al1 other electrophoretic grade chemicals were purchased from Serva.
When the procedure of Heussen and Dowdle [2] was used to determine the sensitivity of TPCK-treated trypsin, a clearly visible activity band was obtained even with only l n g of trypsin (approximately 10 Na-benzoyl-L-arginine ethyl ester (BAEE) mU,Fig. 1A, lane 5). However, the gelatine concentrated in the lower part of the gel during electrophoresis and, due to this effect, the protein markers of 14.4 and 21.5 kDa were hardlyvisible. Using the new procedure, a lower sensitivity to trypsin (10 ng) was obtained (Fig. IB, lane 3). On the other hand, protein bands without protease activity can be visualized easily. The tendency of trypsin to create trails of gelatinolysis was reduced and electrophoretic mobility of proteases was not affected by the presence o f copolymerized gelatine [2]. The main advantage of this method is the possibility to compare directly the gelatinolytic activity pattern with positions of components recognized by specific probes (Fig. 2). When partially purified boar acrosin was analyzed, the distribution o f fucose-bearing sites and positions of immunologically detected acrosin corresponded to gelatinolytic activity. However, other proteolytic activity bands were also observed (Fig. 2, lane 7). In conclusion, the new method for visualization of proteases described here is simple and time-saving and can be performed in parallel with other analytical methods for specific protein detection. Received December 14, 1990
References
Figure 2. Nitrocellulose replica of partially purified boar acrosin, scparated by SDS-PAGE undernonreducing conditions. (1) Colloidal goldstained low molecular weight standards. (2) Partially purified acrosin stained with colloidal gold. (3) Binding of biotinylated UIex europaeus agglutinin to boar acrosin. (4) Control reaction, using the streptavidin peroxidase conjugate only. ( 5 ) Binding ofmonoclonal antibodyACR.2 to acrosin. (6) Control reaction, using ACR.3 monoclonal antibody. (7) Proteolytic activity zones detected on gelatine-coated nitrocellulose.
[ I ] Granelli-Piperno, A. and Reich, F., J . Exp. Med. 1978, 148, 223-234. [2] Heussen, C. and Dowdle, E.B., Anal. Biochem. 1980, 102, 196-202. [3] Siegel, M. S . and Polakoski, K. L., B i d . Reprod. 1985, 32, 713-720. [4] Laemli, U. K., Narure 1970,227, 680-685. [5] Towbin, H., Staehelin,T. and Gordon, J., Proc. Natl. Acad. Sci. USA 1979, 76, 4350-4354. [6] Moeremans, M., Daneels, G. and Mey, J.D., Anal. Biochem. 1985, 145, 315-321. [7] Zelezna, B. and Cechova, D., Hoppe Seylers Z . Physiol. Chem. 1982, 363, 755-162. [XI Peknicova, J. and Moos, J., Andrologia 1990, 22, 427-435.
