NPGBNitrophenol-P-Guanidino Benzoate
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For immunoabsorption, pooled citrate plasma was diluted fivefold in dilution buffer containing NPGB and mixed with Sepharose 4B-coupled MAbs (1 g/L gel) in a buffer:gel ratio of 2:1.
For Western blotting, 6 mL of plasma pool was diluted in dilution buffer containing a proteinase inhibitor mixture of NPGB, aprotinin, and EDTA and recycled several times at 20 [degrees]C overnight through a 100-[micro]L column of Sepharose-4B-coupled anti-uPA clones 5 and 6 (1 g of each per liter of gel), identical to those used for capture in the ELISA.
[5] Nonstandard abbreviations: uPA, urokinase plasminogen activator; prouPA, proenzyme form of uPA; uPAR, uPA receptor; PAL plasminogen activator inhibitor; MAb, monoclonal antibody; PAb, polyclonal antibody; TNP, trinitrophenyl hapten; and NPGB, p-nitrophenyl guanidinobenzoate.
The immobilized plasminogen was treated with 6 ml of 0.5 mM NPGB in 0.01 M Tris-HCI, (pH= 8.0).
In the first experiment, we treated the immobilized plasminogen with the specific inhibitor NPGB (Table 2).
In NPGB acylation of the immobilized plasminogen, the NPGB binds covalently to the potential substrate binding site of plasminogen, thus the active site is incapable of hydrolyzing the substrate.