**5. Conclusion**

Substrate-SDS-PAGE has been described in 1980 by Heussen and Dowdle, 30 years after description of this technique, several and important advances in methodological approaches to unveil biological systems have been achieved, such as automated DNA sequencing and protein identification through mass spectrometry just to cite a few. In spite of this, this simple and inexpensive methodology still provides powerful and unique information about peptidases. In organisms with complete genome sequences, bioinformatic analysis provides rich information on putative peptidases, however, it cannot be ascertained if the ORFs are indeed expressed and active. It is common to observe an elevated number of putative peptidases in the organisms' genome, with a limited number of active peptidases. While in organisms without genome information, this technique allows the detection and assessment of several biochemical characteristics of the enzymes, such as preferable pH, temperature, catalytic type and substrate preference. Finally, two-dimensional zymography coupled with mass spectrometry for protein identification make possible the broader mapping of active proteolytic enzymes present in a protein extract, allowing the detection of distinct isoforms of peptidases differentiated by a single change in one amino acid of a main peptide. Therefore, thirty years after its first description, zymography still is a powerful approach to unveil peptidases.

### **6. References**


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Substrate-SDS-PAGE has been described in 1980 by Heussen and Dowdle, 30 years after description of this technique, several and important advances in methodological approaches to unveil biological systems have been achieved, such as automated DNA sequencing and protein identification through mass spectrometry just to cite a few. In spite of this, this simple and inexpensive methodology still provides powerful and unique information about peptidases. In organisms with complete genome sequences, bioinformatic analysis provides rich information on putative peptidases, however, it cannot be ascertained if the ORFs are indeed expressed and active. It is common to observe an elevated number of putative peptidases in the organisms' genome, with a limited number of active peptidases. While in organisms without genome information, this technique allows the detection and assessment of several biochemical characteristics of the enzymes, such as preferable pH, temperature, catalytic type and substrate preference. Finally, two-dimensional zymography coupled with mass spectrometry for protein identification make possible the broader mapping of active proteolytic enzymes present in a protein extract, allowing the detection of distinct isoforms of peptidases differentiated by a single change in one amino acid of a main peptide. Therefore, thirty years after its first description, zymography still is a powerful approach to

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**Part 5** 

**Temporal Temperature Gel Electrophoresis** 

