In Vivo Cross-Linking Protocol for Pre-Ribosomes

11-10-2010

In vivo cross-linking protocol for pre-ribosomes

This protocol is used for strains expressing proteins tagged with HIS6-TEV-ProtA or HAT-TEV-ProtA

Recent changes in the protocol are marked red

I use the BY4741 strain background because these cells grow very fast in YPD.

Cross-linking in the Stratalinker

1.  Grow yeast in 1L YPD in 5L flasks at 30ºC to an OD600 of 0.5-0.6. You should get roughly 1g of cells per Liter

2.  Spin down cells, wash with 50 ml of ice-cold PBS and weigh the pellet. Resuspend the cell pellet in 1V (v/w) of ice-cold PBS. Spread the suspension on a standard petridish chilled on ice. UV irradiate (254 nm) the cells in the Stratalinker. For Rrp9, 800-1600 mJ/cm2 is sufficient. Shake the cell suspension after each 800 mJ/cm2 blast. Optimize the UV irradiation time for each protein.

3.  Resuspend the cells in 5 ml of PBS, wash the petridish with 10 ml PBS, pool everything and pellet the cells.

4.  Take off the sup and freeze the cells in liquid N2

Cross-linking in the Megatron

1.  Grow 2.5-3L of yeast in 5-6L flasks at 30ºC in minimal media lacking tryptophan to

an OD600 of 0.5.

2.  Aromatic aminoacids will absorb the UV light hence, growing the cells in minimal

medium without tryptophan will help.

3.  Blast the cells in the Megatron for 80-200 seconds (depending on how well your

protein cross-links). Harvest the cells by centrifugation and aliquot cells in three 50

ml falcon tubes. N2 freeze and store at -80ºC.

Lysis

1.  Add 1V of TN150 buffer (~ 1-1.5 ml) and 2-3V of Zirconia beads to the cell pellet (v/w) to the cell. Vortex 1 min, then 1 min on ice. Repeat five times.

2.  Add 3V TN150 buffer / gram cell pellet, vortex, and centrifuge the suspension in the falcon tube for 20 minutes at full speed in the Heraeus centrifuge (4600 x g).

3.  Transfer the supernatant (~ 4.5 ml) to 1.5 ml eppendorf locking cap tubes and spin lysate for 20 minutes at 20000 x g in a microcentrifuge.

Purification

1.  Transfer 0.5 ml of IgG Sepharose slurry to a falcon tube and wash twice with 5 ml of TN150 buffer.

2.  Mix lysate with beads and nutate for 2 hours at 4ºC.

3.  Spin down the beads (pulse in Hereaus centrifuge to 1000 rpm/ 4ºC) and remove most of the supernatant.

4.  Wash the beads twice with 10 ml TN1000 buffer and twice with 10 ml TN150 buffer.

5.  After the last wash step, resuspend the beads in a small volume of TN150 buffer, transfer to an eppendorf tube and remove the remaining buffer.

TEV cleavage

1.  Add 600 µl of TN150 buffer to the beads (no protease inhibitors + 5 mM beta-mercaptoethanol!!).

2.  Add 2 or 12 µl of home-made GST-TEV protease (depending on which batch~ 20-30 units).

3.  Mix by inverting tube.

4.  Incubate 2 hours at 18ºC, make sure the beads remain in suspension.

5.  Spin the eluates through a mobicol column in an eppendorf tube.

Nickel purification (protocol for analysis of intact RNA)

1.  Take 2% (10 µl) of the TEV eluate and Proteinase K treat as described below.

2.  Add 0.4 g of Guanidium HCl to the TEV eluates (~ 0.5 ml of powder; final concentration is 6M), vortex. Final volume should be around 700 µl. I usually just transfer the TEV eluates to a tube containing the Guanidium powder. Vortex well to dissolve the Guanidium. At this stage, you don't really have to keep the tubes on ice anymore. The Guanidium will protect the RNA from degradation.

3.  To each tube, add 27 µl 5M NaCl (final [NaCl] = 300 mM) and 3 µl 2.5M Imidazole (pH 8.0, final concentration = 10 mM).

4.  Add TN150 buffer to a final volume of 750 µl and add to 50 µl of Nickel-NTA beads equilibrated with wash buffer I.

5.  Nutate 2 hours or o/n at 4ºC. I found that o/n binding generally gives higher yields.

6.  Wash nickel beads twice with 750 µl of wash buffer I and twice with 750 µl of wash buffer II.

7.  Elute proteins in 2 x 200 µl wash buffer II containing 150 mM Imidazole (pH 8.0). Don't forget, wash buffer II already has 10 mM Imidazole in it. Incubate each elution step 5 minutes on ice.

8.  Add EDTA to 5 mM (8 µl 250 mM), SDS to 1% (45 µl 10%) and 100 µg of Proteinase K (5 µl 20 mg/ml; FRESH!) to the eluates and incubate for 2 hour at 55ºC.

9.  Add 50 µl of 3M NaAc pH 5.2 and 500 µl Phenol-Chloroform-Isoamylalcohol, vortex and spin 5 minutes in microcentrifuge at room temperature (otherwise SDS will precipitate).

10. Take supernatant, add 20 µg of glycogen and precipitate RNA with 3V EtOH, wash with 70% EtOH and resuspend pellet in 10 µl of RNase free MQ.

Western blot analysis:

1.  Take 5% of (RNase digested) TEV eluate (25 µl) and add 7 µl of 4x NuPAGE loading dye.

2.  TCA precipitate proteins from the Nickel eluates and resuspend pellet in 40 µl of 1x NuPAGE loading dye.

3.  Load samples (input and eluates) on 1.5 mm 4-12% NuPAGE gel.

4.  Do the Western with Open Biosystems rabbit anti-TAP antibody (1:5000).

Buffers:

TN150 buffer:

50 mM Tris-HCl pH 7.8

150 mM NaCl

0.1% NP-40

5 mM beta-mercaptoethanol (add fresh)

TN1000 buffer:

50 mM Tris-HCl pH 7.8

1 M NaCl

0.1% NP-40

5 mM beta-mercaptoethanol (add fresh)

1 x PNK buffer:

50 mM Tris-HCl pH 7.8

10 mM MgCl2

0.5% NP-40

5 mM beta-mercaptoethanol (add fresh)

5 x PNK buffer:

250 mM Tris-HCl pH 7.8

50 mM MgCl2

50 mM beta-mercaptoethanol

(store at -20ºC)

Wash buffer I:

50 mM Tris-HCl pH 7.8

300 mM NaCl

10 mM Imidazole

6M Guanidine-HCl

0.1% NP-40

5 mM beta-mercaptoethanol (add fresh)

Wash buffer II:

50 mM Tris-HCl pH 7.8

50 mM NaCl

10 mM Imidazole

0.1% NP-40

5 mM beta-mercaptoethanol (add fresh)

Elution buffer:

10 mM Tris pH 7.8

50 mM NaCl

150 mM Imidazole

0.1% NP-40

5 mM beta-mercaptoethanol (add fresh)

Other solutions:

2.5 M Imidazole pH 8.0

Proteinase K:

prepare 20 mg/ml solution in water (make fresh)

Sander Granneman 1