170 Experimental Section 0307-4412(95)00165-4 Zymography of extraceilular matrix proteases ANA R QUESADA, IGNACIO FAJARDO, DANIEL RODR[GUEZ-AGUDO, JOSI~ MANUEL PACHON, MIGUEL ANGEL MEDINA Laboratorio de Bioquimica y Biologia Molecular Facultad de Ciencias Universidad de M~laga E-29071 M~laga, Spain Introduction Electrophoretic techniques are widely used in biochemistry laboratories. Most Experimental Biochemistry courses for undergraduate students devote some effort to training students in electrophoresis. They are mostly taught to perform denatur- ating SDS-PAGE or submarine agarose electrophoresis to sepa- rate proteins or nucleic acids by weight. However, electro- phoretic techniques are also potent and convenient for detecting specific enzyme activities by the technique referred to as zymography. Proteolytic activities, and specifically those of extracellular matrix proteases, are easily and conveniently detected by the use of zymographic techniques 1,2 Zymographic techniques are extensively used for the detection of proteinase activities because they display several advantages, including their simpli- city and high sensitivity. Moreover, they allow the identification of the enzyme of interest in a complex mixture of proteinases, and the determination of the molecular weight of latent and active forms of the enzymes due to the processes of denatura- tion-renaturation carried out in the gel. Samples to be tested are submitted to a non-reducing SDS-PAGE; afterwards, protein activities are reconstituted in situ by washing with buffers con- taining the zwitterionic detergent Triton X-IO0. Finally, specific activities are detected in situ or with the use of an overlaid gel. In this report, we describe the use of zymography to detect type IV collagenases and other matrix metalloproteases, as well as plasminogen activators. We also offer some cheap alterna- tives to train students in zymography. Experimental Conditioned media and cell extracts To prepare conditioned media, cells such as fibroblasts are grown in six-well plates. When subconfluent, medium was aspirated, cells were washed twice with phosphate-buffered saline and each well received 1 ml of culture medium without serum and with 200 KIU of Trasylol. Cells were washed twice with phosphate-buffered saline and extracted by using 0.2% Triton X-100 in 0.2 M Tris- HCI. Both conditioned media and cell extracts were centrifuged for 10 min at 1000 g and supernatants were stored at -20°C until used. In duplicate samples, cells were trypsinized and counted. Gelatin zymography Conditioned media and cell extracts were submitted without previous heating to SDS-PAGE in the cool room, as described by Laemmli, 3 with the following modifica- tions. Reducing agent (DTI' or mercaptoethanol) was absent and the resolving gel contained gelatin (1 mg/ml). After electro- phoresis, the gel was washed twice in 50 mM Tris-HCl, pH 7.4, supplemented with 2% Triton X-100, and twice with 50 nM Tris- HCI (pH 7.4). Each washing step was carried out for 10 min at room temperature and with agitation. Afterwards, the resolving gel was covered with a substrate buffer (composition: 50 mM Tris-HCl, 5 mM CaC12, 1% Triton X-100, 0.902% Na3N, pH 7.4) and incubated overnight at 37°C. Finally, the gel was stained with a fixing mixture containing Coomassie Blue R for 30 min and destained with the same mixture without the dye. Casein zymography The procedure was as described for the gelatin zymography but with casein (0.5 mg/ml) instead of gelatin in the resolving gel. As previously shown, 4 a pre-run of the casein gel is required. Plasminogen activator zymography In this case, there was no addition to the resolving gel. After electrophoresis and washing, a substrate gel (composition: 2.7% skimmed dry milk, 0.8% agar, 0.0005% Na3N and plasminogen 0.44 KUI/ml in phosphate buffered saline) was laid over the resolving gel. The system was left overnight in a humidified chamber at 4°C, and then it was incubated in the humidified chamber at 37°C until lytic bands were detectable on a dark field, Activation with APMA Extracellular matrix proteases are activ- able by treatment with 1 mM p-aminophenyl mercuric acetate (APMA) for lh at 37°C. Suggestions for a practical on zymography for undergraduated students. The procedures described are easy to carry out and can be performed by students in a two-day session. In the first day, students are provided with the theoretical background, and they carry out the electrophoresis, and washing. In the second day, they carry out the staining and destaining, they observe the results and discuss them. In those labs with access to culture cell facilities, students should be provided with conditioned media and/or cell extracts from one or several cell lines. However, this requirement is not a limitation and some cheap alternatives are possible for those labs without access to culture cell facilities. In fact, some inex- pensive commercially available proteases could substitute for the conditioned media. They include trypsin, pepsin or the col- lagenase extracted from Clostridum histo~yticum (reference C0-7926 in the Sigma catalogue). When students are provided with commercial proteases, they could be asked to prepare suc- cessive dilutions from a stock in order to determine an optimal concentration for the easy observation of the proteolytic activity. For instance, in the case of Clostridum histolyticum collagenase a dilution series between 100 /~g/ml and 100 pg/mol would be convenient. In the same way, an alternative to conditioned media for plasminogen zymography could be a serial dilution of the commercially available urokinase. Results In both casein and gelatin zymographies, proteolytic bands are observed as unstained bands in an otherwise stained resolving gel. In plasminogen zymography, the lysis band is observed as a dark band surrounded by clear casein gel when observed on a dark field. We have successfully used these zymographic tech- niques in our studies of extracellular proteases produced by cancer cells, 5 including neuroblastoma, rhadomyosarcoma, osteosarcoma, and mammary carcinoma cells. A convenient, easily maintained and non-hazardous cell line is Ehrlich ascites carcinoma. Clearly observable bands are obtained with media conditioned by Ehrlich cells and Ehrlich cell extracts in both gelatin-zymograms (two main bands, corresponding to 92 and 72 kDa gelatinases; see Figure 1) and casein-zymograms (a band, corresponding to the 92 kDa gelatinase, which also exhibits BIOCHEMICAL EDUCATION 24(3) 1996