Zymography, Casein Zymography, and Reverse Zymography: Activity Assays for Proteases and their Inhibitors
Proteases and their inhibitors play an important role in the extracellular matrix (ECM) remodeling that occurs in a number of physiological and pathological processes. These include such diverse processes as trophoblast implantation, mammary gland involution, rheumatoid arthritis and tumor cell invasion (Liotta et al. 1991a; Liotta et al. 1991b). Matrix metalloproteinases involved in ECM degradation (Birkedal-Hansen et al, 1993; Stetler-Stevenson et al., 1993) have been found to be subject to regulatory controls at multiple levels including transcription, mRNA stability, translation, secretion, activation of proenzymes, degradation and inhibition by specific endogenous inhibitors known as Tissue Inhibitors of Metalloproteinases or TIMPS (Matrisian, L.M., 1992; Stetler-Stevenson et al, 1993b). The need to rapidly and easily screen tissue extracts, biological fluids or the conditioned media of a number of cell types has resulted in the adoption of the zymogram technique in many laboratories. The reverse zymogram is a related technique which detects metalloproteinase inhibitors. These techniques utilize electrophoresis for the separation of the various protease and inhibitory species coupled with the detection of the activity within the polyacrylamide gel. These techniques are simple to perform, highly sensitive in detecting secreted protease and protease inhibitory activity, and flexible enough to be utilized for a variety of proteases and their inhibitors.
KeywordsConditioned Medium Ammonium Persulfate Plasminogen Activator Activity Mammary Gland Involution Gelatin Zymogram
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