reverse-phase hplc hp 1090 ii liquid chromatograph  (Gilson Inc)

Journal: Scientific Reports

Article Title: Loss of the major Type I arginine methyltransferase PRMT1 causes substrate scavenging by other PRMTs

doi: 10.1038/srep01311

Figure Lengend Snippet: Cell pellets from PRMT1 wild-type and knockout MEFs were acid hydrolyzed and the resulting amino acids separated by high-resolution cation exchange chromatography as described in "Methods". The separation of standards (1 μmol ) of ADMA, SDMA, and MMA/arginine with ninhydrin detection as described by Zurita-Lopez et al . (2012) is shown in the control chromatograph (a). The separation of these amino acids is typical, although small changes in the elution times can occur between runs. Cell hydrolysates were then chromatographed without standard amino acids and fractions analyzed by reverse-phase HPLC after derivatization with OPA for fluorescence quantification as described in "Methods". HPLC conditions were optimized to separate the large pool of arginine from ADMA and SDMA in wild-type (b) and PRMT1 knockout (c) and from MMA in wild-type (d) and PRMT1 knockout (e) samples . The total amount of a given species was quantified by summing the integrated area under the curve for all HPLC fractions containing the respective species that are consistent with the migration on the cation-exchange column.

Article Snippet: After incubating the mixture at room temperature for 200 s, 5 μL of 0.75 M HCl was added and the sample was vortexed by hand for 5 s. The resulting fluorescent isoindole derivatives were separated and quantified using reverse-phase HPLC (HP 1090 II liquid chromatograph coupled to a Gilson Model 121 fluorometer with excitation and emission filters of 305–395 nm and 430–470 nm, respectively, and a setting of 0.01 RFU).

Techniques: Knock-Out, Chromatography, Control, Fluorescence, Migration