Wittrup: Cell Surface Secretion Assay

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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:
    1. Resuspend cells in 20 μl 10mg/mL avidin dissolved in PBS and incubate at room temperature for 20 minutes.
    2. 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.