Overlay zymography fibrin

The following section gives a brief description of the materials and methods required to perform fibrin overlay zymography. For more comprehensive and illustrative details, the reader is referred to the accompanying references. 

The following section demonstrates the usefulness of fibrin overlay zymography for the detection of PAs and PAIs in complex biological systems. Despite the generally qualitative nature of overlay zymography, semiquantitative results may be obtained by densitometric analysis of PA/PAI calibration standards appropriate to the specific application. 

Fig. 3. Densitometric analysis of plasminogen activators. Various concentrations of urokinase-type plasminogen activator (uPA) and tissue-type plasminogen activator (tPA) were elec-trophoresed on 10% polyacrylamide gels and subjected to fibrin overlay zymography. The indicator gels were densitometrically scanned as described (M3). Data is shown as mean with error bars indicating standard deviation (SD). Linear regression analysis was performed and correlation coefficient (r) is shown (r = 1.000, perfect correlation).

Fig. 5. Reverse fibrin zymography for detection of PAIs. The fibrin overlay zymogram was supplemented with uPA (see Section 3.6). Samples 1-5 were from conditioned media of bovine corneal endothelial cells at different growth stages and in the presence of various PA inducers (X2). Increased amido black staining indicating position of PAI-1 (55-kDa) is shown (lanes 1 and 2). A small amount of uPA (45-kDa) and tPA (70-kDa) in lanes 3-5 is also noted.

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