The substrate-protease pair is coexpressed at stoichiometric amounts inside a 2A-based bicistronic vector, amenable to genetic manipulation
The substrate-protease pair is coexpressed at stoichiometric amounts inside a 2A-based bicistronic vector, amenable to genetic manipulation. assay can probe PCSK9 proteolysis under conditions of either genetic or molecular perturbation inside a high-throughput manner. This system is definitely well suited for both the biochemical evaluation of clinically found out missense single-nucleotide polymorphisms (SNPs), as well as for the screening of small-molecule inhibitors of PCSK9 proteolysis. E. colistrain will reduce incubation instances, but any cloning strain will become suitable. Plate the transformations onto Luria-Bertani (LB) agar comprising 50 – 100 g/mL carbenicillin. Incubate the plates over night at 37 C. Select 2 – 4 colonies to grow inside a small-scale tradition (2 – 5 mL of LB medium with 50 – 100 g/mL carbenicillin). Incubate the tradition at 37 C at 220 rpm until it is turbid. Isolate the plasmid DNA from your cells using a plasmid DNA purification (for 5 min to recover cells. Aspirate trypsin-containing medium and reconstitute the cells in the tradition medium to a concentration of 2 x 105 cells/mL. Using a multichannel pipette, transfer 100 L of the cells to each well of a white (opaque-bottom) 96-well plate, which gives a final seeding concentration of 2 x 104 cells/well. Notice: For initial experiments, it may be useful to additionally seed a sister, clear-bottom 96-well plate, so as to monitor cell growth and adherence during the protocol and guidebook long term troubleshooting. Incubate the cells at 37 C and 5% CO2 for 24 h. Prepare a master plate of plasmids inside a 96-well format. Dilute each plasmid in elution buffer (Tris-HCl, pH 8.5) to 50 ng/L in an individual well of a 96-well plate. Prepare 4 wells each for the positive control (WT) plasmid8, bad control (S386A) plasmid8, and plasmid-free buffer (for mock transfections). If drug or additional cellular-based treatments are being planned, then prepare the expert plate with the positive control (WT) plasmid in each well, along with 4 wells each of the bad control (S386A) plasmid and the plasmid-free buffer. Transfection (day time 1) Prepare the transfection combination inside a 96-well plate file TCS 21311 format using deep-well 96-well plates. Perform the transfections in triplicate, being sure to prepare plenty of reagents to account for pipetting and transfer deficits. Note: The following calculations prepare enough reagent to run one 96-well plate in triplicate (estimated conservatively for 420 wells). Help to make a master mix of a diluted lipid transfection reagent, adding 50.4 L of reagent to 2050 L of reduced-serum medium. Make TCS 21311 a expert mix of a DNA precomplexation reagent, adding 33.6 L of reagent into 1730 L of reduced-serum medium. Using a multichannel pipette, aliquot 16.8 L of the diluted DNA precomplexation reagent mixture into MIF each well of the deep-well plate. Using a multichannel pipette, aliquot 3.2 L (160 ng) of each plasmid from your master plate into each well of the deep-well plate. Using a multichannel pipette, aliquot 20 L of the diluted lipid transfection reagent combination to each well of the plate and blend the contents of each well using the multichannel pipette. Cover the plates and let them sit at room temp (RT) for 10 TCS 21311 – 15 min to form lipid:DNA complexes. Notice: Upon transfection, the final parts per well will become as follows: 40 ng of DNA, 0.12 L of transfection reagent, and 0.08 L of DNA pre-complexation reagent, and the content of each well will be 10 L in total volume (reduced-serum medium). Softly exchange the medium within the 293T cells in the 96-well plates using a multichannel pipette, taking care not to disrupt the cells. Replace the aspirated medium with 95 L of DMEM supplemented with 10% FBS. Notice: This would be an appropriate time to treat the cells with any drug of interest. Add 10 L of the transfection combination to each appropriate well a multichannel pipette. Softly swirl the plate to mix the material in the wells. Incubate the plate at 37 C with 5% CO2 for 24 h. Notice: The final volume of the wells comes to 105 L, to account for evaporation over 24 h. Assay (day time 2) Prepare a stock remedy for coelenterazine reagents: 3 M sodium ascorbate [dissolved in phosphate-buffered saline (PBS), prepared refreshing], 5 M NaCl, 10 mg/mL bovine serum albumin (BSA; dissolved in PBS, prepared refreshing), and 2 mM coelenterazine (dissolved in acidified methanol comprising 200 L of 3 N HCl per 10 mL). Notice: The 2 2 mM coelenterazine can be stored for 2 weeks when it is kept at -80 C and in the absence of light. Prepare 2x coelenterazine reagents for the luciferase readout, with a separate reagent each for the cells and medium, relating to Table 5. Blend all reagents save the coelenterazine 1st, filter the combination through a 0.22-m syringe filter, and.