TX-100 FRACTIONATION PROTOCOL

If tissue stored at -80°C, place in -20°C for at least 4h 0m 0s or 4h 0m 0s to thaw prior to homogenization.

Prepare 1X TNE with phosphatase and protease inhibitors.

Weigh tissue out in mg.

Add in 10 volumes of 1X TNE.

1. 10µL of TNE per 1mg of tissue.
2. Ex: 50mg tissue = 500µL of 1X TNE.

Either by mechanical (Dounce) or homogenizer machine, homogenize tissue gently and On ice.

This is TNE crude lysate (no detergents).

Take specific volume of tissue in TNE and add in equal volume of 1X TNE w/ 2% Triton X-100 (Tx100).

• Ex. 150µL of TNE tissue + 150µL 1X TNE+2% Tx100.

Sonicate @ 4°C.

3 pulses:0h 0m 10s ON / 0h 0m 2s OFF.

Spin down.

Option 1 : 16000x g,4°C.

Option 2 : 20000x g,4°C.

After spin:

Supernatant = soluble . Save supernatant and add equal volume of complete TNE (cTNE).

1. Sonicate @ 4°C: 3 pluses: 0h 0m 10s ON / 0h 0m 2s OFF.
2. Boil: 0h 10m 0s at 95°C.
3. Spin down: 16000x g,4°C.
4. Supernatant from this is the soluble fraction .

Pellet = insoluble .

Wash pellet in 150µL of 1X TNE+1% Tx100.

Resuspend pellet via pipette.

Spin down.

1. Option 1 : 16000x g,4°C.
2. Option 2 : 20000x g,4°C (25000x g,0h 0m 0s) .

Resuspend pellet in 75µL to 100µL of cTNE.

Sonicate @ 4°C: 3 pluses: 0h 0m 10s ON / 0h 0m 2s OFF.

Boil: 0h 10m 0s at 95°C.

Spin down: 16000x g,4°C.

Supernatant from this is the insoluble fraction .