Wittrup: Cell Surface Secretion Assay

Cell Surface Secretion Assay
Andy Rakestraw May 5, 2006

Cell Labeling

 * 1) Induce protein expression for ten to twelve hours by growth in SG-CAA or YPG. Remove 1 OD600 of cells.
 * 2) 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.
 * 3) 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.
 * 4) Incubate cells at room temperature for thirty minutes vortexing every ten minutes (keep in dark if labeling with fluorescein).
 * 5) Wash cell three times with 1 mL PBS/0.1%BSA.
 * 6) For biotin labeling:
 * 7) Resuspend cells in 20 μl 10mg/mL avidin dissolved in PBS and incubate at room temperature for 20 minutes.
 * 8) 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.

Plate Preparation

 * This step can be done while the cells are being labeled.


 * 1) 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.
 * 2) 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.
 * 3) 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.
 * 4) 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

 * 1) 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.)
 * 2) Collect in a 50 mL conical and spin down cells (3500 rpm for 5 minutes.
 * 3) 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.