The protocol is provided as an example for a 6-well plate format. Amounts for other plate sizes are given in Table 1.
- Set up cells to be transfected so they are approximately 0.6 x 105-4.0 x 105 cells per well (approximately 70-80% confluent) at the time of transfection.
- Add 0.5-5 µl of protein to a sterile tube containing the appropriate amount of serum-free medium.
- Add 3 µl of TransPass P Transfection Reagent (mix well before use).
- Gently mix the transfection complex mixture by flicking the tube.
- Incubate at room temperature for 20 minutes.
- Remove serum-containing growth media from cells by aspirating, wash cells with serum-free medium and add 1 ml of serum-free medium to each well.
- Add the transfection complex mixture to cells.
- Return plate to incubator and incubate for 2-5 hours.
- Add 1 ml of complete media (containing 10% serum) to each well.
- Replace media on the following day and continue incubation until assaying. Wash cells with serum-free medium before assaying to remove any untransfected protein.
* The transfection complex mixture is composed of protein and TransPass P Transfection Reagent in serum-free medium. For example, at the incubation step (step 8) (6-well format), transfection complex mixture consisting of 2 µg protein, and 3 µl TransPass P Transfection Reagent in 200 μl serum-free medium is added to a well containing cells in a 1 ml volume.