Preparation of nuclear extract and cytoplasmic extract

 

Solutions:

Buffer A (Hypotonic Buffer): 1L
10 mM HEPES pH 7.9 10 ml 1M HEPES, pH 7.9
1.5 mM MgCl2 1.5 ml 1M MgCl2
10 mM KCl 3.33 ml 3M KCl
0.5 mM DTT 500 µl 1M DTT
0.2 mM PMSF 1 ml 0.2 M PMSF

Buffer B (S100 extraction buffer): 500 ml
0.3 M HEPES pH 7.9 150 ml 1M HEPES, pH 7.9
1.4 M KCl 233.3 ml 3M KCl
30 mM MgCl2 15 ml 1M MgCl2

Low Salt Buffer: 1L
20 mM HEPES pH 7.9 20 ml 1M HEPES, pH 7.9
25% Glycerol 250 ml Glycerol
20 mM KCl 6.67 ml 3M KCl
1.5 mM MgCl2 1.5 ml 1M MgCl2
0.2 mM EDTA 400 µl 0.5 M EDTA
0.5 mM DTT 500 µl 1M DTT
0.2 mM PMSF 1 ml 0.2 M PMSF

High Salt Buffer: 1L
20 mM HEPES pH 7.9 20 ml 1M HEPES, pH 7.9
25% Glycerol 250 ml Glycerol
1.2 M KCl 400 ml 3M KCl
1.5 mM MgCl2 1.5 ml 1M MgCl2
0.2 mM EDTA 400 µl 0.5 M EDTA
0.5 mM DTT 500 µl 1M DTT
0.2 mM PMSF 1 ml 0.2 M PMSF

Buffer D (Dialysis Buffer): For 2 L
20 mM HEPES, pH 7.9 1 M HEPES 7.9 40 ml
20 % Glycerol Glycerol 400 ml
100 mM KCl 3 M KCl 66.67 ml
0.2 mM EDTA 0.5 M EDTA 800 µl
0.5 mM DTT 1M DTT 1 ml
0.2 mM PMSF 0.2 M PMSF 2 ml

 

Splicing Extract Preparation

1. Resuspend cell pellet in ~5 packed cell volumes of cold PBS. Split into additional 250 ml conical tubes if necessary. Spin at 1500 rpm for 10 minutes in J-6.

2. Resuspend the washed cell pellet in 5 packed cell volumes Buffer A (Hypotonic Buffer). Spin at 1500 rpm for 5 minutes in J-6.

3. Add Buffer A up to a final of 3 original packed cell volumes (i.e., add ~2 volumes [do not exceed a total of 3 volumes]). Incubate on ice for 10 minutes.

4. Transfer the cells to a Wheaton A Dounce homogenizer and lyse with ~10 strokes. Stain an aliquot with Trypan Blue and check for >90% lysis on microsope.

5. Spin at 2000 rpm for 15 minutes in J-6 to pellet nuclei.

6. Save the supernatant for S100 extract (see below).

7. Transfer the nuclear pellet to a glass beaker containing a stir bar and add 1/2 packed nuclear volume of Low Salt Buffer.

8. While stirring gently, add 1/2 packed nuclear volume of High Salt Buffer (1.2 M KCl). Homogenize in Dounce if necessary.

9. Stir for 30 minutes in cold room.

10. Transfer the nuclear extract to 30 ml Corex tubes and spin at 10,000 rpm for 30 minutes in HB-4 rotor at 4°C.

11. Remove the supernatant and dialyze for ~2 hours in >50 volumes Buffer D in cold room. Change buffer and dialyze against >50 volumes for ~2.5 hours.

12. Transfer to 30 ml Corex tubes and spin at 10,000 rpm for 20-30 minutes in HB-4 rotor at 4°C.

13. Aliquot in 1 ml aliquots and freeze in liquid nitrogen. Store at -80°C.

S100 extract

A. Mix cytoplasmic extract with 0.11 volumes buffer B.

B. Spin at 39,400 for 60 minutes in Oakridge tubes in Ti70 rotor at 4°C.

C. Dialyze for ~2 hours then ~2.5 hours in >50 volumes Buffer D lacking PMSF in cold room.

D. Spin 14,000 rpm for 30 minutes in SS-34 rotor in Oakridge tubes.

E. Aliquot S100 into 1 ml fractions and freeze on N2 and store at -80°C.