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Are there some additional instructions for how to run the HPLC analysis for the S4U Incorporation assay (e.g. Column, temperature, injection volume, and flow rate)?

The samples should be run on a C18 column. In principle any C18 column could be used instead of the Supelco Discovery C18 reverse phase column (size 250 x 46 mm, particle size 5 mM), as indicated in the SLAMseq Explorer and Kinetics Kits User Guide. Column oven temperature to use is 30 °C, with an injection volume of 100 µl, and flow rate of 500 µl/min. With these settings the run time is around 70 minutes. It is important to wash the column between runs. For additional details of standards, mobile phase solutions and isocratic gradient to use please refer to the SLAMseq Explorer and Kinetics Kits User Guide (p.16-17).

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