Wittrup: Cell Surface Secretion Assay
Cell Surface Secretion Assay
Andy Rakestraw May 5, 2006
- Induce protein expression for ten to twelve hours by growth in SG-CAA or YPG. Remove 1 OD600 of cells.
- Wash three times in 500 μl of carbonate buffer pH 8.4 (4.2% NaHCO3 and 0.034% NaCO3)-carbonate buffer only good for two weeks at 4°C.
- Resuspend cells in 10 μl 100 μg/μl NHS-PEG-fluorescein (or NHS-PEG-biotin) diluted into carbonate buffer. Solution will be very viscous and will need frequent vortexing.
- Incubate cells at room temperature for thirty minutes vortexing every ten minutes (keep in dark if labeling with fluorescein).
- Wash cell three times with 1 mL PBS/0.1%BSA.
- For biotin labeling:
- Resuspend cells in 20 μl 10mg/mL avidin dissolved in PBS and incubate at room temperature for 20 minutes.
- Wash as in PBS/BSA as described above and resuspend in the biotinylated-protein of your choice. For D1.3 capture 30 μl of biotin-lysozyme was used. Incubate for 20 minutes at room temperature and wash as described above.
- This step can be done while the cells are being labeled.
- Make solution of 30 wt/v% polyethylene glycol (8 kDa) and YPG/BSA. (This solution is 50% PEG by volume. i.e. use 5 ml of PEG powder and then add YPG to total of 10 ml of solution.) You can accelerative dissolution by incubating solution at 42°C.
- Filter sterilize the media. You will need ~9mL for every 2 OD600 of cells. Add labeled and washed cells to YPG/PEG (2 OD/9mL as described) and apply 9 mL of suspension to 10x10 cm square sterile petri dish.
- Rock dish back and forth until the bottom is completely covered. Get rid of any large bubbles by aspirating them with a pipet. (Getting rid of bubbles is important as they will eventually pop and cause the media to recede to the middle.
- Incubate in a 30°C incubator for an empirically determined amount of time (~11 hours for the alpha mating factor screening experiments).
Cell Washing and Labeling
- After sufficient incubation, wash cells with 10 ml PBS/BSA. (If your secreted protein has a fast off-rate, you may want to add competitor to the wash buffer. This addition is not necessary for 4m5.3 secretion.)
- Collect in a 50 mL conical and spin down cells (3500 rpm for 5 minutes.
- Wash in 1 mL PBS/BSA and transfer to 1.5 ml tube. Wash twice more in 1 mL PBS/BSA and then label with antibodies similarly to surface display.