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. 2013 Mar 6;8(3):e58087. doi: 10.1371/journal.pone.0058087

Figure 1. Analysis of recombinant CD28 expression in HEK cells.

Figure 1

(A) Coimmunoprecipitation. Recombinant HEK cells were lysed with lysis buffer, and 200–500 µl of cell lysate was incubated with rabbit αFLAG antibody at 4°C for 2 hours, then 20 µl of protein A agarose slurry (GE Healthcare) was added for another 2 hours. The beads were washed three times with at least 10 volumes of lysis buffer before resolving by SDS-PAGE. Detection was done either with mouse αFLAG or mouse αCD28. As control HEK293-SLP2-FLAG was used. 1: HEK293 lysate, 2: HEK293-CD28-FLAG lysate, HEK293-SLP2-FLAG lysate. (B) Westernblot. Cells were lysed and analysed by immunoblot using αFLAG or αCD28 antibodies. 1: HEK293 lysate, 2: HEK293-CD28-FLAG lysate (C) Elisa. Recombinant CD28 is recognized by a commercial αCD28 mAb. HEK293-CD28-FLAG lysate is coated on NUNC maxisorp via FLAG-tag. Detection was done with 1: αCD30 or 2: αCD28.