2-Methoxy-2,4-diphenyl-3(2H)-furanone-labeled gelatin zymography and reverse zymography: a rapid real-time method for quantification of matrix metalloproteinases-2 and -9 and tissue inhibitors of metalloproteinases.
Measurement of matrix metalloproteinases (MMPs) and their specific tissue inhibitors of metalloproteinases (TIMPs) by the techniques of zymography and reverse zymography provide useful information regarding the status of matrix accumulation or breakdown. This report describes the use of 2-methoxy-2,4-diphenyl-3(2H)-furanone (MDPF), a fluorescent compound which can be used to label gelatin as a substrate for detection of the gelatin degrading MMP-2 and -9 by zymography. In addition, a modification of the zymographic technique by addition of excess MMPs enables the use of the MDPF-labeled gelatin substrate for the identification and quantification of TIMPs by reverse zymography. Both systems are real-time sensitive reliable quantification techniques, easily used for measurement of these MMPs and TIMPs in clinical, biological, and tissue culture samples.
Min
Danqing
D
Department of Endocrinology, Royal Prince Alfred Hospital, Camperdown, NSW, Australia. danqingm@med.usyd.edu.au
Lyons
James Guy
JG
Jia
Junhong
J
Lo
Lisa
L
McLennan
Susan V
SV
D000818:Animals; D002460:Cell Line; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D004847:Epithelial Cells / Q000201:enzymology; D005456:Fluorescent Dyes / Q000737:chemistry; D005663:Furans / Q000737:chemistry*; D005780:Gelatin / Q000737:chemistry*; D006801:Humans; D008264:Macrophages / Q000201:enzymology; D020778:Matrix Metalloproteinase 2 / Q000378:metabolism*; D020780:Matrix Metalloproteinase 9 / Q000378:metabolism*; D051381:Rats; D019714:Tissue Inhibitor of Metalloproteinases / Q000378:metabolism*; D016212:Transforming Growth Factor beta / Q000494:pharmacology
D005456:Fluorescent Dyes; D005663:Furans; D019714:Tissue Inhibitor of Metalloproteinases; D016212:Transforming Growth Factor beta; C008394:2-methoxy-2,4-diphenyl-3(2H)-furanone; D005780:Gelatin; D020778:Matrix Metalloproteinase 2; D020780:Matrix Metalloproteinase 9