Gelatin zymography is widely used to detect and evaluate matrix metalloproteinase-9 (MMP-9) activity. MMP-9 transcription was previously shown to be negatively regulated by ganglioside GDla. [D. Hu, Z. Man, T. Xuan, P. Wang, T. Takaku, S. Hyuga, X.S. Yao, T. Sato, S. Yamagata, T. Yamagata, Ganglioside GDla regulation of matrix metalloproteinase-9 (MMP-9) expression in mouse FBJ cell Lines: GD1a suppression of MMP-9 expression stimulated by PI3K-Akt and p38 though not by the Erk signaling pathway, 2006, submitted for publication.] Zymography of MMP-9 of FBJ-M5 cells preincubated with GD1a indicated a greater decrease in activity than expected from mRNA suppression. Incubation of conditioned medium containing MMP-9 with GDla caused MMP-9 activity to decrease. Examination was thus made to confirm that MMP-9 activity is actually suppressed and/or MMP-9 protein undergoes degradation by GDla. GDla was found to have no effect on MMP-9 activity and Western blots indicated GDla not to diminish MMP-9 during electrophoresis under reducing conditions. GDla appeared to mediate the binding of a portion of MMP-9 with certain molecules, with consequently greater molecular mass on the gel, to cause decrease in the activity of MMP-9 at the site where it would normally appear. Caution should be used in doing gelatin zymography since molecules other than GDla may similarly work, causing decrease in MMP-9 activity in zymography. (c) 2006 Elsevier Inc. All rights reserved.