Trypsin digestion

This is a protocol optimised to digest mitochondria isolated from cultured cells (HEK293) with trypsin

1.      For a strong (loss of integral mitochondrial proteins) digestion:  1 µg trypsin at 1µg/µl for 10 µg of mitochondrial extracts. For a weak digestion (loss of mitochondrial proteins facing the cytosol): 1 µg trypsin at 1µg/µl for 1 mg of mitochondrial extracts.

2.      Isolate mitochondria and dose the quantity of mitochondrial proteins isolated. Do not freeze them and process them (points 5-9) immediately after dosage.

3.      Prepare a stock dilution of trypsin: resuspend the powder (Trypsin for Bovin Pancreas ; Sigma ; T1426-50mg) to obtain a stock solution of 5µg/µl in HCl. For long-term storage keep this solution at -20°C

4.      Prepare a working solution of trypsin at 1µg/µl in H₂O right before the digestion.

5.      Use 30µg of mitochondrial proteins and digest for at least two timepoints: 10 min (short digestion) and 30 min (longer digestion). Add intermediate points if needed.

6.      To digest mitochondria, add the trypsin on ice and incubate this solution at 37°C for the time required (see above), then immediately place the samples back on ice.

7.      Stop the digestion by adding laemli sample buffer 4X

8.      Boil the samples for 5 min at 95°C

9.      Freeze the samples at -20°C.

10. Proceed with SDS-PAGE