This paper reports the experimental application of isoelectric focusing coupled with isotachophoresis on a networked microfluidic chip, which has an ITP channel and an IEF channel. Those two channels share a channel segment, where the transition from IEF to ITP takes place. A mixture of three fluorescent proteins was first concentrated and segregated into adjacent ITP zones. After the ITP zones migrated into the channel segment, current was switched from ITP channel to IEF channel. A pH gradient was established by ampholytes pre-filled in the IEF channel and the proteins in the segment were separated by IEF. More IEF channels will be added along the ITP channel to separate protein samples in the ITP train in future. As the future work is completed, protein will be first segregated by ITP according to electrophoretic mobility and then separated by IEF based on pI.