pCEP-4-MBP

Protocol tips

Upstream tips	Protocol tips	Downstream tips
HEK293 cells were seeded at 90% confluency in a 24-well plate and allowed to adhere overnight.	Next day, cells were transfected with different constructs in triplicates using FuGENE® HD (Promega, Mannheim, Germany, Cat. no.: E2311). FuGENE® HD was added to DMEM/F-12, GlutaMAX™ (Life Technologies, Darmstadt, Germany, Cat. no: 10565–018) at RT for 10 min and then incubated with different plasmid DNA for additional 20 min. The transfection reagent to plasmid DNA ratio was 1:3 (0.5 μg plasmid per well). The different transfection DNA mixtures were directly added to the respective wells and incubated for 24 h.

Upstream tips
HEK293 cells were seeded at 90% confluency in a 24-well plate and allowed to adhere overnight.
Protocol tips
Next day, cells were transfected with different constructs in triplicates using FuGENE® HD (Promega, Mannheim, Germany, Cat. no.: E2311). FuGENE® HD was added to DMEM/F-12, GlutaMAX™ (Life Technologies, Darmstadt, Germany, Cat. no: 10565–018) at RT for 10 min and then incubated with different plasmid DNA for additional 20 min. The transfection reagent to plasmid DNA ratio was 1:3 (0.5 μg plasmid per well). The different transfection DNA mixtures were directly added to the respective wells and incubated for 24 h.

Publication protocol

HEK293 cells were seeded at 90% confluency in a 24-well plate and allowed to adhere overnight. Next day, cells were transfected with different constructs in triplicates using FuGENE® HD (Promega, Mannheim, Germany, Cat. no.: E2311). FuGENE® HD was added to DMEM/F-12, GlutaMAX™ (Life Technologies, Darmstadt, Germany, Cat. no: 10565–018) at RT for 10 min and then incubated with different plasmid DNA for additional 20 min. The transfection reagent to plasmid DNA ratio was 1:3 (0.5 μg plasmid per well). The different transfection DNA mixtures were directly added to the respective wells and incubated for 24 h. On the next day the supernatant was discarded and fresh serum free medium was added for additional 24 h before the supernatant or cells were harvested for western blot analysis. To detect membrane bound proteins cells were lysed using the cOmplete Lysis-B EDTA-free Kit according to the manufacturing guidelines (Roche, Cat. no.: 04719948001).

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