Egg Extract Preparation

1. Female X. laevis.

2. Two- to three-gallon plastic buckets.

3. Wide-bore plastic Pasteur pipets.

4. Beckman ultracentrifuge rotor SW41 and 12-mL tubes (14 x 89 mm, Ultraclear No.344059), or SW55 rotor and 5-mL tubes (13 x 51 mm, Ultraclear No. 344057).

5. Pregnant mare serum gonadotropin (PMSG; Calbiochem No. 367222): Stable for 2 wk at -20°C in deionized water (DW).

6. Human chorionic gonadotropin (Sigma cat. no. CG-10): Stable for 2 wk at 4°C in DW.

7. 10X Marc's modified Ringer's solution (MMR): 1 M NaCl, 20 mM KCl, 10 mM MgSO4, 20 mM CaCl2, 1 mM EDTA, 50 mM HEPES. Adjust to pH 7.8 with NaOH. Autoclave to sterilize.

8. 2% (w/v) L-Cysteine hydrochloride monohydrate (Sigma, cat. no. C7880): Adjust to pH 7.7 with KOH. Must be prepared fresh.

9. Cycloheximide (Sigma, cat. no. C7698): Blocks protein synthesis including cyclin B and keeps the extract in S phase.

10. Lysis buffer stock solution: 250 mM sucrose, 50 mM KCl, 2.5 mM MgCl2,10 mM HEPES (pH 7.8). Sterilize by filtration and store at 4°C.

11. Lysis buffer: Add 1 mM dithiothreitol (DTT) and 50 |g/mL cycloheximide to the stock solution. Must be prepared fresh.

12. Cytochalasin B (Sigma No. C6762): 5 mg/mL in dimethyl sulfoxide (DMSO). Store at -20°C.

13. DTT (Sigma, cat. no. D9779): 1 M in DW. Store at -20°C.

14. Aprotinin (Sigma A1153): 5 mg/mL in DW. Store at -20°C.

15. Leupeptin (Sigma L2884): 5 mg/mL in DW. Store at -20°C.

2.2. Nuclear Remodeling Assay

1. Tissue culture cells. We routinely use Xenopus embryonic fibroblast cells XL2.

2. Cell dissociation solution (Sigma, cat. no. C5914): Store at 4°C.

3. Digitonin (Calbiochem, cat. no. 300410): 10 mg/mL in DMSO.

4. Bovine serum albumin (BSA), fraction V, protease free (Roche 100350): 10% stock solution in DW.

5. Spermidine trihydrochloride (Sigma, cat. no. S2501): 10 mM in DW.

6. Spermine tetrahydrochloride (Sigma, cat. no. S2876): 10 mM in DW.

7. Pepstatin A (Sigma P5318): 2 mg/mL in methanol.

8. Phenylmethylsulfonyl fluoride (PMSF) (Sigma, cat. no. P7626): 100 mM in 2-propanol. Store at 23°C.

9. Transplantation buffer: 250 mM sucrose, 75 mM NaCl, 0.5 mM spermidine trihydrochloride, 0.15 mM spermine tetrahydrochloride, 15 mM HEPES (pH 7.8). Add 1 mM DTT, 1 |ig/mL leupeptin, 2 |ig/mL pepstatin A, and 0.1 mM PMSF just before use. Spermidine and spermine will maintain nuclear integrity. pH is 7.3 to 7.5 without adjustment.

10. Mix 0.1 M ATP and 0.1 M GTP in 10 mM HEPES (pH 7.8).

11. Phosphocreatine (Sigma, cat. no. P6502): 0.4 M in 10 mM HEPES (pH 7.8).

12. Creatine phosphokinase (Sigma, cat. no. C3755): 5 mg/mL in 10 mM HEPES (pH 7.8) and 50% glycerol.

Fig. 1. Sucrose cushion apparatus. Set the poly-L-lysine-coated cover slip on top of the white bottom piece (left) with the coated side upward and overlay the top piece (middle) on them. Insert the two pieces into a Falcon 2059 tube carefully not to break the cover slip (right).

13. Energy regeneration system (ERS): Combine 1 vol of the mixture of 0.1 M ATP and 0.1 M GTP, and 5 vol of 0.4 M phosphocreatine. After mixing well, add 2 vol of 5 mg/mL creatine phosphokinase. Prepare fresh.

14. Apyrase grade VII (Sigma, cat. no. A6535). 125 U/mL in 10 mM HEPES (pH 7.8).

15. All components should be kept at -20°C unless indicated otherwise.

2.3. Sucrose Cushion Method for Immunocytochemistry

1. 12-mm Round cover slips (Fisher, cat. no.12-545-80).

2. Apparatus for sucrose cushion (Medical Specialties, Baltimore, MD; Fig. 1).

3. Falcon 14-mL tubes No. 2059.

4. Poly-L-lysine (Sigma cat. no. P5899): 1 mg/mL phosphate-buffered saline (PBS). Store at -20°C.

5. 4% Paraformaldehyde in PBS.

6. 30% (w/v) sucrose in PBS: Filter to sterilize and store at 4°C.

7. Histology pen (DAKO S2002).

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