Facile Identification and Isolation of Protease Using SDS-PAGE and Zymography

Purification and identification of novel proteases is critical in the development of new therapeutic drugs, such as those for ischemic stroke. However, it's a time-consuming and expensive job to purify enzymes from natural sources. Furthermore, even if the process is completed, the enzyme could be a known protease. Zymography is a protease-confirming assay, but its molecular weight is hard to be accurately measured due to background noise and the broad zone of lysis. Moreover, a sample in a zymography gel cannot be used for protein identification because of the staining substrate in the gel. However, a protease in an unstained SDS-PAGE gel is not damaged by staining buffer and its proteolytic potential will be maintained and be identified, even though these bands are invisible. In this research, to take advantage of both zymography and unstained SDS-PAGE, each band in an unstained SDS-PAGE gel was cut and transferred to a zymography gel. Through this method, bands on the SDS-PAGE gel and the accompanying electroblotted membrane could be used to confirm the target proteolytic activity. Screened samples could be used for protease identification and to check for novelty.