Figure 1

Schematic illustration of the TIMP activity assay. The steps involved in TIMP activity assay during incubation, SDS treatment (inactivates MMPs, their reactivation is accomplished by SDS removal after electrophoresis) and zymography, respectively. (A): Symbol explanation and schematic illustration, (B): schematic zymogram and (C): a true zymogram. In lane 1, α₂M, represented in a monomeric form (180 kDa), is visible as a high molecular weight protein band at the gel top. In lane 2, activity from active MMP-2 is visible as a lysis band at 64 kDa. Lane 3 illustrates the basic principle of the assay: active MMP-2 cleaves the α₂M bait region during incubation, is entrapped and stays covalently entrapped after SDS treatment. This is evident by a lysis band at the top of the zymogram together with α₂M. In lane 4, MMP-2 is pre-incubated with TIMP before the adding of α₂M. During this pre-incubation TIMP will bind to MMP-2 and inhibit enzyme activity. When α₂M is thereafter added, MMP-2 cannot cleave the bait region. The following SDS treatment disassociates TIMP from MMP-2, and MMP-2 reappears on the zymogram as a 64 kDa lysis band. In lane 5, TIMP-1 is pre-incubated with MMP-1EA (TIMP-binding capacity but without enzymatic activity) which results in TIMP-1 capture. When MMP-2 and α₂M are thereafter simultaneously added, MMP-2 is active and free to cleave the α₂M bait region resulting in entrapment. SDS disassociates the TIMP/MMP-1EA complex but the α₂M-MMP-2 complex stays intact. On the zymogram only the α₂M-MMP-2 complex is visible at the gel top.