Methods for Overlay Zymography

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 fibrin indicator gel was prepared as outlined in the schematic (Fig. 1) and as described previously (R4, R5). Briefly, plasminogen-rich fibrinogen was slowly solubilized at a concentration of 5mg/ml in 0.85% (w/v) saline (prewarmed to 37 °C) with gentle mixing (inversion several times over 1.5-2 h) in a temperature-controlled water bath at 37 °C. Slow solubilization of fibrinogen was necessary to prevent protein flocculation. Agarose was separately prepared as a 1% (w/v) solution in phosphate-buffered saline (PBS, pH 

For detection of plasminogen activator inhibitors (PAIs), uPA was incorporated into the indicator agarose gel at a concentration of 0.8 U/ml (El, R4). Under these conditions, lysis occurs everywhere except where inhibitor has difiused from the polyacrylamide gel into the agarose indicator gel. Thus, lytic-resistant zones such as PAIs will appear as dark bands against a cleared background (i.e., reverse zymography). 

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. 

Fibrin zymograms may be subjected to densitometric analysis to obtain quantitative information about PA activity (Fig. 3). As can be seen, good calibration curves were obtained for both uPA and tPA over an approximately 

---