A method for activity staining after native polyacrylamide gel electrophoresis using a coupled enzyme assay and fluorescence detection: application to the analysis of several glycolytic enzymes.
We describe a method for the detection of isoforms of several glycolytic enzymes by activity staining after native PAGE. The staining is based on coupled enzyme assays carried out on the gel after electrophoresis and is linked to the disappearance of NADH, which is visualized by fluorescence. This method offers reliable and sensitive detection for phosphoenolpyruvate carboxylase, PPi-dependent phosphofructokinase, and pyruvate kinase from plant tissues. It can be applied to the detection of all enzymes which are normally detected spectrophotometrically using coupled enzyme assays consuming NAD(P)H.
Rivoal
Jean
J
Department of Plant Science, University of Manitoba, Winnipeg, Manitoba R3T 2N2, Canada. rivoalj@ms.umanitoba.ca
Smith
Christopher R
CR
Moraes
Trevor F
TF
Turpin
David H
DH
Plaxton
William C
WC
D029688:Brassica napus / Q000201:enzymology; D000460:Chlorophyta / Q000201:enzymology; D004796:Clinical Enzyme Tests / Q000379:methods; D003979:Diazonium Compounds / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D005453:Fluorescence; D010730:Phosphoenolpyruvate Carboxylase / Q000032:analysis*; D025221:Phosphofructokinases / Q000032:analysis*; D011770:Pyruvate Kinase / Q000032:analysis*; D012680:Sensitivity and Specificity; D013050:Spectrometry, Fluorescence / Q000379:methods; D013053:Spectrophotometry / Q000379:methods; D013194:Staining and Labeling / Q000379:methods*
D003979:Diazonium Compounds; C067037:Fast Violet B; D025221:Phosphofructokinases; D011770:Pyruvate Kinase; D010730:Phosphoenolpyruvate Carboxylase