Assessment of caspase-4 released free AFC by RP-HPLC and fluorescence detection.
A simple RP-HPLC method based on fluorescence detection was developed for the quantitation of 7-amino-4-trifluoro methylcoumarin (AFC) in cell lysates from JEG-3 choriocarcinoma cells for determination of caspase-4 activity. In contrast to the established methods of AFC detection using a fluorescence microplate reader or using a fluorescence photometer, the separation of AFC-signals from interfering fluorescence signals by a reversed phase column affords more precise quantitation of released AFC. This can be important for analyses of cell lysates with low caspase activity or experimental series with marginal differences among samples. By applying this new method, a linear dynamic range of 40pmol/mL to 3nmol/mL with a correlation coefficient of 0.9996 was achieved. Due to the short retention time ( approximately 7min), the determination of AFC by RP-HPLC under isocratic conditions requires small amounts of samples (50microL injection volume), and allows increased sample throughput. This method should be easily applied with little or no modification to other caspase assays by using the same fluorophore.
Koehn
Sandra
S
Friedrich-Schiller-University Jena, Department of Obstetrics, Placenta-Labor, Bachstrasse 18, 07743 Jena, Germany.
Trueck
Mike
M
Poehlmann
Tobias G
TG
Schleussner
Ekkehard
E
Markert
Udo R
UR
Seyfarth
Lydia
L
D053471:Caspases, Initiator / Q000378:metabolism*; D045744:Cell Line, Tumor; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D003374:Coumarins / Q000032:analysis / Q000378:metabolism; D006801:Humans; D015203:Reproducibility of Results; D013050:Spectrometry, Fluorescence / Q000379:methods*; D013379:Substrate Specificity
D003374:Coumarins; C055467:7-amino-4-trifluoromethylcoumarin; C093516:CASP4 protein, human; D053471:Caspases, Initiator