Protein expression experiments in mammalian cells
Protein expression experiments in mammalian cells
Content from Compact Molecular Biology Laboratory Guide (5th Edition)
Operation method
Basic program Transient expression with COS cells
Materials and Instruments
Carrier (e.g. CDM 8) Move 1. Subclone the target gene into a suitable vector to obtain the desired recombinant DNA. Purify the recombinant DNA using either a small volume method (5 ml culture medium) or centrifugation with CsCl/ethidium bromide. 2. 2. The day before transfection, seed COS-7 cells at approximately 20% confluence into 100 mm dishes containing DMEM-2 CS medium (so that the cells can grow to approximately 50% confluence the next day). Allow the cells to grow overnight to approximately 50% sinks. 3. 3. Prior to use (for each COS cell in a 100 mm dish to be transfected), mix 5 ml of DMEM-2 NS culture medium at 37°C thoroughly with 0.2 ml of DEAE-Dextran/Chloroquine solution, and then add 5-10 recombinant DNA. 4. Aspirate the culture medium of COS cells and add DMEM-2 CS/DEAE-dextran/DNA prepared in step 3 to each 100 mm dish. incubate the cells for 3~4 h. Observe the cells with differential phase microscope. DEAE-dextran will cause cells to shrink and become vacuolated. Prolonged incubation may increase the transfection efficiency, while on the other hand, it may cause cell death. 5. Aspirate DMEM/DEAE-dextran/DNA and add 5 ml 10% DMSO (prepared with PBS). Incubate the cells at room temperature for 2 min. 6. 6. Aspirate out the DMSO and add 10 ml of DMEM-2 CS. allow the cells to grow overnight (12-20 h). If a large number of cells float after overnight incubation, start again, but the cells should be grown for one more day in the second step. 7. 7. Pass the transfected COS cells from each 100 mm dish to two new 100 mm dishes. After transfection, COS cells do not grow well, and passaging the cells the next day helps to restore growth and increase protein expression levels. In addition, DEAE-dextran-treated cells become sticky, and the next day's passaging restores the wall-adherent characteristics of the cells so that they may float again after gentle treatment with PBS and EDTA. 8.1 When secreted proteins are expressed 96 h after completion of step 7, add 5 ml of DMEM-2 CS and incubate for 4 days. Harvest the culture medium and centrifuge at ~1000 g for 10 min at room temperature to remove dead cells and debris and retain the supernatant. Detect secreted proteins by biological identification or metabolic labeling and immunoprecipitation, immunoaffinity chromatography, radioimmunoassay or immunoblotting. 8.2 When cell surface proteins or intracellular proteins are expressed 72 h after transfection according to step 6, aspirate the culture medium from the cells. Add 5 ml of PBS, shake to wash, and aspirate the PBS. add 5 ml of 0.5 mmol/L EDTA dissolved in PBS, and incubate for 15 min. gently remove the cells from the culture dish with a Pasteur pipette. The cells were gently removed from the culture dish with a Pasteur pipette. The cell surface proteins were stained with a suitable fluorescent antibody and detected by microscopy or flow cytometry. Caveat Unless otherwise specified, all cells were incubated at 37°C, 6% CO2humidified incubator. For more product details, please visit Aladdin Scientific website.