pcDNA3.1-MRP4-his6

Protocol tips

Upstream tips	Protocol tips	Downstream tips
HEK293T cells were seeded in a six-well plate with 300,000 cells/well in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin 24 h prior to transfection.	For transfection, 4 µg of plasmid DNA was combined with 18 µL of 10 mM linear polyethylenimine (PEI; Polysciences, Warrington, PA) and 100 µL of reduced serum media (OPTIMEM) and added to each well. Twenty-four hours after transfection the media was replaced with DMEM containing 10% FBS and 1% penicillin/streptomycin. Samples were taken every 24 h over a 72 h period.

Upstream tips
HEK293T cells were seeded in a six-well plate with 300,000 cells/well in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin 24 h prior to transfection.
Protocol tips
For transfection, 4 µg of plasmid DNA was combined with 18 µL of 10 mM linear polyethylenimine (PEI; Polysciences, Warrington, PA) and 100 µL of reduced serum media (OPTIMEM) and added to each well. Twenty-four hours after transfection the media was replaced with DMEM containing 10% FBS and 1% penicillin/streptomycin. Samples were taken every 24 h over a 72 h period.

Publication protocol

pcDNA3.1-MRP4-his6 plasmid was constructed by restriction digestion of MRP4-his6 out of the pFastBac plasmid and ligation into a pcDNA 3.1 Zeo + plasmid. pOPINE-MRP4–3C-flag-his8 was made by the Oxford Protein Production Facility (OPPF, Harwell, UK). pcDNA3.1-MRP4 without a his-tag was a kind gift from Professor Susan Cole (Queen’s University, Kingston, ON, Canada). HEK293T cells were seeded in a six-well plate with 300,000 cells/well in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin 24 h prior to transfection. Three hours prior to transfection, the media was replaced with low-serum DMEM containing 2.5% FBS and 1% penicillin/streptomycin. For transfection, 4 µg of plasmid DNA was combined with 18 µL of 10 mM linear polyethylenimine (PEI; Polysciences, Warrington, PA) and 100 µL of reduced serum media (OPTIMEM) and added to each well. Twenty-four hours after transfection the media was replaced with DMEM containing 10% FBS and 1% penicillin/streptomycin. Samples were taken every 24 h over a 72 h period.

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Manufacturer protocol

Download the product protocol from Alice J. Rothnie, Life & Health Sciences, Aston University for pcDNA3.1-MRP4-his6 below.

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