Water Saturated 1-butanol Recipe For Chicken

Fig. 4. (A) SDS-polyacrylamide gel of Sir2-Af2 with 10-fold dilutions showing more than 99% purity. (B) SDS-polyacrylamide gel of TmSir2p with 10-fold dilutions showing approximately 99% purity. Arrowheads indicate position of 30 kDa marker.

Basic Protocol for Expression and Purification ofSir2-Tm

1. Inoculate 1 liter of glucose-enriched M9ZB medium with 10 ml of overnight growth of BL21 codon+ (DE3) (Stratagene) in a selective medium of carbenicillin (0.1 mg/ml).

2. Grow the cells at 37° in carbenicillin (0.1 mg/ml). When the OD66o = 0.5-0.6, induce the cells with a 1 mM final concentration of IPTG. Induce for 4-6 hr at 37°.

3. Spin the cells at 5000 rpm for 30 min at 4° in an RC-3B Sorvall centrifuge and discard the supernatant carefully.

4. Resuspend the pellet in 50 ml of lysis buffer Tm and run the cells through a microfluidizer or French press. Spin the lysate at 13,000 rpm for 30 min at 4° in a Sorvall GSA or similar rotor and carefully discard the supernatant.

5. Wash the pellet by resuspending completely in 100 ml of lysis buffer Tm and spin at 12,000g for 30 min at 4°.

6. Resuspend the washed pellet in 100 ml of lysis buffer Tm, spin at 760g for 10 min at 4°, and collect inclusion bodies in the supernatant. Spin the supernatant at 12,000g for 30 min at 4° to collect inclusion bodies in the pellet.

7. Wash the inclusion bodies five times by resuspending them in 100 ml of lysis buffer Tm, and spinning at 12,000g for 30 min at 4°. After a final wash resuspend them again and spin at 760g for 10 min at 4°, discard the pellet, and spin the supernatant at 12,000g for 30 min at 4° to collect inclusion bodies in the pellet.

8. After the final spin, solubilize the inclusion bodies in 5 ml of buffer E by rocking them in a tube overnight at 4°. Determine the protein concentration and dilute in buffer E to a final concentration of 1 mg/ml or less. Renature the protein by dialyzing into 0 M urea, keeping everything else in the buffer E constant.

9. Dialyze the protein into buffer C and run over a Q-Sepharose Fast Flow ion-exchange column (Pharmacia). Elute the protein in a salt gradient with buffer C-NaCl.

10. Optional: Dialyze protein fractions in buffer F and run through a MonoQ 10/10 anion exchange column (Pharmacia) and elute the protein in a salt gradient with buffer C-NaCl.

11. Dialyze the protein in buffer D, run it through a Superdex-75 sizing column, and collect fractions of pure protein (see Fig. 4B).

Basic Protocol for Purification of Saccharomyces cerevisiae Sir2p and Homo sapiens Sir2A Expressed as N-Terminal Fusions to Glutathione S-Transferase Using pGEX Vectors

1. Inoculate 50 ml of Luria-Bertani (LB) medium supplemented with ampicillin (100 /xg/ml) with DH5a containing Sir2 expression vector and grow overnight at 37°.

2. Inoculate 500 ml of LB medium supplemented with ampicillin (lOO/xg/ml) with 5 ml of overnight culture and grow at 30° until the culture reaches an OD600 of about 0.7.

3. At that point induce with 100 /¿MIPTG and transfer the culture to a 20° incubator. Grow overnight.

4. In the morning pellet the cells in a GS3 Sorvall rotor (4000 rpm, 10 min,

5. Wash the cells once with cold phosphate-buffered saline (PBS).

6. Resuspend the cell pellet in PBS supplemented with 0.5 mM dithiothreitol (DTT), 0.5 mM phenylmethylsulfonyl fluoride (PMSF), and protease inhibitors.

7. Disrupt the cells by French press.

8. To the cell suspension add NaCl to increase the final salt concentration to 350 mM. Sir2 from S. cerevisiae has a tendency to precipitate during subsequent steps of purification, so a final concentration of 350 mM NaCl is maintained in the buffers used during the purification.

9. Spin the cell suspension in an ultracentrifuge equipped with a in Ti70 rotor (Beckman, Fullerton, CA) at 40,000 rpm for 1 hr at 4°.

10. Fill a disposable column (Bio-Rad, Hercules, CA) with an appropriate volume of glutathione-Sepharose 4B beads (Pharmacia).

11. Equilibrate the column with 10 volumes of PBS.

12. Apply the supernatant.

13. Wash the column with 20 volumes of 50 mM Tris-HCl (pH 8.0), 350 vaM NaCl.

14. Elute the protein from the column with 10 volumes of 50 mM Tris-HCl, 350 mM NaCl, 20 mM reduced glutathione (Pharmacia).

15. Dialyze the eluant against 2 liters of storage buffer [25 mM HEPES-NaOH (pH 8.0), 200 mM NaCl, 20% (v/v) glycerol], concentrate, and store in small aliquots.

Buffers and Solutions for Purifications

Glucose-enriched M9ZB medium M9 minimal salt 1 x solution NaCl: final concentration, 5 g/liter

Tryptone or Casamino Acids (Difco Laboratories, Detroit, MI): 10 g/liter Glucose (added after autoclaving): 0.4% (w/v) MgS04 (added after autoclaving): 2 mM

Lysis buffer Af HEPES (pH 7.0), 20 mM EDTA, 2 mM DTT, 1 mM Pefabloc, 0.1 mg/ml Aprotinin, 5 /zg/ml Leupeptin, 2 /u,g/ml

Buffer A Bis-Tris (pH 6.0), 20 mM DTT, 1 mM ZnCl2 25 ¡jlM

Buffer A-NaCl Bis-Tris (pH 6.0), 20 mM DTT, 1 mM ZnCl2, 25 ixM NaCl, 1 M

Buffer B Bis-Tris propane (pH 8.5), 20 mM DTT, 1 mM ZnCl2, 25 ¡iM

Buffer C Tris (pH 8), 40 mM DTT, 1 mM ZnCl2, 25 ßM

Buffer C-NaCl Tris (pH 8), 40 mM NaCl, 1 M DTT, 1 mM ZnCl2, 25 ßM

Buffer D HEPES (pH 8), 20 mM NaCl, 150 mM DTT, 1 mM ZnCl2, 25 iiM

Lysis buffer Tm: Keep at 4° Tris (pH 7.5), 50 mM EDTA, 1 mM NaCl, 100 mM

Buffer E

Tris (pH 8), 50 mM Urea, 4 M NaCl, 100 mM DTT, 1 mM ZnCl2, 25 ¡iM

Buffer F

Tris (pH 8), 40 mM NaCl, 100 mM DTT, 1 mM ZnCl2, 25 fiM

Purification of Acetylated Chicken Histones as Histone Deacetylase Substrate

Chicken histones used for labeling with [3H] acetic acid are isolated from the blood of white Leghorn chickens injected with 1% (w/v) phenylhydrazine.

Chickens were fasted for 48 hr, followed by daily injections of 1% (w/v) phenyl-hydrazine in 10 mM sodium phosphate buffer, pH 7.2, for 7 days. Birds were killed and blood was collected with heparinized syringes.

1. Spin 10 ml of chicken blood at lOOOg for 5 min at room temperature.

2. Resuspend the cells in 50 ml of Swim's S-77 medium (Sigma, St. Louis, MO).

3. Spin the cells (lOOOg, 5 min, room temperature) and wash them with 50 ml of the same medium one more time.

4. Resuspend the cells in 100 ml of Swim's S-77 medium supplemented with 10% (v/v) newborn calf fetal serum and 10 mM sodium butyrate.

5. Add 10 mCi of [3H]acetic acid (9.4 Ci/mmol; ICN, Costa Mesa, CA) and incubate at 37° for 30 min.

6. Spin the cells at lOOOg for 5 min at room temperature and resuspend them in 100 ml of Swim's S-77 medium supplemented as described above and incubate for 1 hrat37°.

7. Cool the cells on ice and spin them at 3000g for 5 min at 4°.

8. Resuspend the cells in 20 ml of NIB buffer (see Media Required for Purification of Acetylated Chicken Histones, below, for buffer and solution recipes) and incubate for 30 min on ice.

9. Spin at 3000g for 5 min at 4° and wash twice in 20 ml of NIB buffer.

10. After a final spin (3000g, 5 min, 4°), resuspend the pellet in 20 ml of Tris-NaCl solution.

11. Add 2.3 ml of 4 M sulfuric acid and incubate on ice for 1 hr.

13. Precipitate with 10 volumes of ice-cold acetone-5 M HC1 (99:1, v/v).

15. Lyophilize the pellet and resuspend in 5 ml of 20 mM Tris-HCl (pH 6.0), 10 mM sodium butyrate.

16. Clarify the slurry by centrifugation at 8000g for 10 min at 4°, aliquot the supernatant, and freeze at —80°.

This labeling procedure yields histones with specific activity between 700 and 1300 cpm//zg.

Media Required for Purification of Acetylated Chicken Histones

NIB buffer:

20 mMpiperazine-M/V'-bis(2-ethanesulfonic acid) (PIPES, pH 6.8), 0.25 M sucrose, 60 mM KC1,5 mM MgCl2,1 mM CaC^, 10 mM sodium butyrate, 1 mM PMSF, 0.5% (v/v) Triton X-100

Tris-NaCl solution:

Histone Deacetylation Assays

The assays are done in a final volume of 100 /xl. Each reaction contains the following:

NaCl, 50 mM

3H-labeled chicken erythrocyte histones, 13,000 cpm//xg

1. The reaction mixture is incubated at 30° for S. cerevisiae Sir2, at 37° for human Sir2A, and at 55° for A. fulgidus Sir2.

2. The reaction is stopped by adding 36 /xl of 1 M HC1,0.4 M acetic acid and extracted with 0.8 ml of ethyl acetate with 10 min of incubation on ice.

3. Organic phase (600 /xl) is added to 3 ml of scintillation fluid and the samples are counted in a scintillation counter.

NAD+ Measurements from Intact Yeast Cells

1. Inoculate 50-ml cultures in appropriate growth medium, and shake overnight at 30°.

2. Measure the Agoo of the overnight culture and dilute to an A^oo of 0.2 in a final volume of 500 ml in a 1-liter flask. Continue shaking the culture at 30° and allow it to grow to an A6oo of approximately 1.0. The protocol can be scaled down.

3. Transfer an equal number of cells for each culture into a 500-ml centrifuge bottle (on ice). Example: If a 450-ml culture is at an A^oo of 1.0, take 400 ml of a culture that is at an Aeoo of 1.125. Balance the bottles with fresh medium.

4. Pellet the cells in the GS3 Sorvall rotor at 2500 rpm for 10 min at 4°. Discard the supernatant down the sink.

5. Resuspend each of the cell pellets with 40 ml of ice-cold water by vortexing and pour the mixtures into 50-ml conical (blue cap/Falcon) tubes (again on ice).

6. Pellet the cells in an Eppendorf swinging-bucket centrifuge at 2500 rpm for 5 min at 4°.

7. Discard the supernatant down the sink and resuspend the cells in 5 ml of 1 M formic acid saturated with butanol (on ice) (see Buffers Needed, below, for recipe). Mix by swirling, not by vortexing. Incubate on ice for 30 min, swirling at three equally spaced intervals to prevent the cells from settling to the bottom of the tubes.

8. Transfer the mixtures by pouring into 30-ml Oakridge centrifuge tubes (on ice).

9. Add a 1/4 volume (1.25 ml) of ice-cold 100% trichloroacetic acid (TCA) and mix by swirling. Incubate on ice for 15 min.

10. Pellet the cells in the SS-34 rotor at 6000 rpm (4000g) for 20 min at 4°.

11. Transfer the supernatants to 50-ml Falcon tubes, using a 10-ml glass pipette. Try to avoid taking any cell debris. (Note: The NAD+ is in this fraction.) Store on ice. In the case of the mock culture, avoid taking the lower liquid phase.

12. Wash the pellets with 2.5 ml of ice-cold 20% (w/v) TCA. (Vortex to resuspend the pellets.)

13. Pellet the cells again in the SS-34 rotor at 6000 rpm for 20 min, 4°.

14. Carefully remove the supernatant with a 5-ml glass pipette and combine with the first TCA supernatant in the 50-ml Falcon tube.

15. Spin the tube containing the supernatants in an Eppendorf tabletop centrifuge at 3000 rpm for 5 min at 4°. This will pellet any remaining cell debris.

16. Set up alcohol dehydrogenase (ADH) reactions:

Water 338 fil

Ethanol, 100% 2 /A Alcohol dehydrogenase (15-mg/ml stock in 50 mM Tris, pH 7.5) 10 fxl

Acid extract from cells 150 ¡i\

Mix each tube by briefly vortexing and incubate in a 30° water bath for 20 min.

17. Using a single quartz cuvette, blank the spectrophotometer at 340 nm with the mock extract reaction without ADH added, and then read the A340 for each sample.

Buffers Needed

Formic acid (1 M) saturated with butanol (250-ml stock): Combine 212 ml of water, 13 ml of formic acid [88% (w/v) stock], and 25 ml of 1-butanol; mix and allow the phases to separate, and then bring the volume up to 250 ml with water and top off with 1-butanol. Store at 4°.

ADH buffer (2x) Tris (pH 9.7), 0.6 M Lysine-HCl, 0.4 M

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