Nuclear and Cytoplasmic Extracts

Materials:

• Endonuclease (Sigma E8263)

• 10% Triton X or IGEPAL 630

• Phosphatase inhibitor Solution

• Phosphate buffered Saline (PBS), pH 7.4

• Solution A (see recipe at the end of protocol)

• Solution B (see recipe at the end of protocol)

• Solution C (see recipe at the end of protocol)

• DeStreak Rehydration Solution (Amersham-Biosciences)

Method:

1. Wash cell plate with ice-cold PBS. Add 1ml ice-cold PBS with Phosphatase inhibitor

solution per 10cm plate and scrape cells. Transfer to an ice-cold Eppendorf tube.

2. Pellet cells at 1000 X g for 2 minutes. Remove supernatant.

3. Add Solution A without resuspending the pellet at a 1:1 (v/v) ratio to that of the

cellular pellet. Centrifuge as in step 2. Remove supernatant.

4. Add Solution A at a 1.5:1 volume of Solution A per volume of cell pellet. Briefly vortex

or resuspend pellet, incubate on ice for 10 minutes. Add 1/20th volume [cell pellet +

Solution A] of 10% Triton X (or IGEPAL 630) to a final concentration of 0.5% and mix.

Centrifuge 6000 rpm @ 4 ºC for 30 seconds.

5. Collect supernatant (cytoplasmic extract). Determine concentration, aliquot into 200 μg samples and store immediately at –80 ºC.

6. Add Solution B in a 1:1 volume relative to nuclear pellet and gently resuspend.

Centrifuge 12,000 rpm @ 4 ºC for 10 minutes. Discard supernatant.

7. Add DeStreak Rehydration solution in a 2:1 ratio to the pellet. Vortex for 5

minutes at room temperature.

8. Add endonuclease (Sigma, E8263) to a final concentration of 300 units/ml and

incubate at room temperature for 30 minutes.

9. Sonicate the sample for 10 seconds. Centrifuge 13k rpm @ 4 ºC for 30 minutes.

Collect supernatant (nuclear extract). Determine protein concentration.

10. Aliquot into 200 μg samples and store immediately at –80 ºC.

Reagents and Solutions:

A. Solution A (low salt buffer)

Note:

*DTT and PMSF should be added immediately prior to use

1. Add M.Q. H₂O to a final volume of 20 ml.
2. First add DTT then PMSF and quickly vortex vigorously
3. Keep the solution on ice and store at 4°C

B. Solution B (low salt sucrose buffer)

Note:

*DTT and PMSF should be added immediately prior to use

1. Add 6.8 grams sucrose (final concentration 1.0 M)
2. Add M.Q. H₂O to a final volume of 20 ml.
3. First add DTT then PMSF and quickly vortex vigorously
4. Keep the solution on ice and store at 4°C

C. Solution C (High salt buffer)

Note:

*DTT and PMSF should be added immediately prior to use

1. Add M.Q. H₂O to a final volume of 2 0ml
2. Add DTT first then PMSF and quickly vortex vigorously
3. Keep the solution on ice and store buffer at 4°C

D. Phosphatase Inhibitor Salt Solution

50 X Preparation. Dilute to 1 X in Phosphate Buffered Saline (1X PBS)

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