Transient protein expression experiments
Transient protein expression experiments
Three factors make the COS cell expression system suitable for high-level, short-term expression of proteins: (i) the plasmid containing the SV40 replication start site reaches a high copy number in cells 43 hours after transfection; (ii) there are many good late-cell expression/penetration vectors available; and (iii) a variety of easy-to-use methods for efficiently transfecting COS cells have been established.
Operation method
basic program
Materials and Instruments
Carrier Move 1. Subclone the target gene into a suitable vector to obtain the desired recombinant DNA, and purify the recombinant DNA by the small volume method (5 ml culture) or by centrifugation with CsCl/ethidium bromide. For more product details, please visit Aladdin Scientific website.
Bovine serum DMEM Dextran PBS DMSO EDTA
Petri dishes Incubator Phase contrast microscope Centrifuge
2. COS-7 cells grown to confluence in DMEM-10 CS (approx. cells per 100 mm dish) were divided into flasks at a ratio of 1:5 on the day before transfection so that on the next day the cells would have grown to approximately 50% confluence, and the cells were allowed to grow to approximately 50% confluence in a CO2 incubator at 37°C overnight.
3. Prior to use (corresponding to each 100 mm dish of COS cells to be transfected) mix 55 ml of DMEM-10 NS culture medium at 37°C thoroughly with 5 to 10 μg of recombinant DNA, then add 0.2 ml of dextran/chloroquine solution and mix well.4. Aspirate the culture medium of COS cells and add DMEM-10 CS/DEAE-dextran/DNA prepared in step 3 to each 100 mm flat blood. incubate the cells in a CO2 incubator at 37°C for 3-4 h (may need to be optimized), and observe the cells with a phase contrast microscope.5. Aspirate DMEM/DEAE-dextran/DNA, add 5 ml of 10% DMSO (prepared in PBS), and incubate the cells at room temperature for 2 min. Aspirate DMSO and add 10 ml of DMEM-10 CS. Allow the cells to grow in the incubator at 37°C overnight (12~20 h).
6. Transfer the transfected COS cells from each 100 mm dish to two new 100 mm dishes. Warm the cells at 37°C according to step 7a or 7b.7a. When secreted proteins were expressed, 96 h after completion of step 6, 5 ml of DMEM-10CS was added and cultured for 4 days. The culture was harvested and centrifuged at ~1,000 g for 10 min at room temperature to remove dead cells and debris, and the supernatant was retained for detection of secreted proteins by metabolic labeling, immunoprecipitation, immunoaffinity folding, radioimmunoimmunoassay, immunoblotting, or biological identification.7b. When expressing cell surface proteins or intracellular proteins, 72 h after transfection according to step 5, aspirate the culture medium from the cells, add 5 ml of PBS, shake and wash, and aspirate the PBS. 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 hold at 37℃ for 15 min in a COS incubator, gently remove the cells from the dish with a Pasteur pipette, and then stain the cell surface proteins with a suitable fluorescent antibody, and then detect them by microscopy or flow cytometry.