APEX proximity labeling reactions

From strains validated to express active APEX:

• Quench exponentially proliferating cells with 100 μg/mL cycloheximide.

• Collect cells by centrifugation and wash with freezing buffer (15% glycerol, 150 mM potassium acetate, 2 mM magnesium acetate, 20 mM HEPES/sodium hydroxide pH 7.2, 0.5% (w/v) glucose, 100 μg/mL cycloheximide).

• Resuspend in the same freezing buffer (1E+08 cells in 60 μL buffer), and store at -80 °C until further use.

• For each labeling reaction, thaw 1E+08 cells on ice, and wash in 1 mL 0.1M MES/sodium hydroxide pH 6.5, 100 μg/mL cycloheximide.

• Resuspend in 0.2 mL of this buffer containing 0.01% digitonin (1 μL added from a 20 mg/mL digitonin stock in DMSO, stored at -80 °C).

• Incubate in a 30 °C shaking water bath for 30 min. 

• Collect cells by centrifugation,  and wash with 1 mL of ice-cold 1.2M sorbitol/PBS solution .

• Resuspend in 0.2 mL of 1.2M sorbitol/PBS containing 2.5 mM phenol-biotin (1 μL added from a 0.5M stock in DMSO, stored at -80 °C).

• Incubate on ice for 90 min.

• About 15 min before the end of the 90 min incubation on ice, prepare a quenching solution by mixing the following: 20 μL Trolox (from a 0.5M stock in DMSO, stored at -80 °C); 20 μL sodium azide (from a 1M stock in water, stored at -80 °C); 0.2 mL of a 10 mM sodium ascorbate solution in PBS, prepared fresh.

• Also before the end of the 90 min incubation on ice, prepare a 0.2M stock of hydrogen peroxide (by dilution of the 30% (9.8M) hydrogen peroxide solution;  stored at 4 °C).

• At the end of the 90 min incubation on ice, expose the cells to 2mM hydrogen peroxide (2 μL were added to the 200 μL cell suspension from the 0.2M solution).

• Vortex briefly, and incubate on ice for 2 min.

• Stop labeling by adding 0.2 mL of the freshly prepared quenching solution described above.

• Proceed for immunoblotting or IP, to analyze and isolate the biotinylated proteins.