Isolation of gTuRC

Buffers and solutions:

 

HEPES Homogenization Buffer:                           H100:

50 mM K-HEPES, pH 7.6                                        50 mM K-HEPES, pH 7.6

100 mM KCl                                                              100 mM KCl

1 mM MgCl2                                                             1mM MgCl2

1 mM K-EGTA, pH 7.6                                            1 mM K-EGTA, pH 7.6

10% glycerol

1:100 protease inhibitors

1:100 PMSF

 

30% PEG-8000 in H100

 

20% IPEGAL (detergent that replaces NP40)

 

H200:

50 mM K-HEPES, pH 7.6                                                                                       

200 mM KCl

1 mM MgCl2                                                            

1 mM K-EGTA, pH 7.6

 

I make H100 and H200 by mixing stocks of H0 (same buffer but without KCl) + H1 (same buffer but with 1M KCl):         

 

For 10 ml H200, + additives, no detergent:  2 ml H1M, 8 ml H0, 2 ml 0.5M GTP,             0.7 ml b-mercaptoethanol, 10 ml protease inhibitors.

 

For 25 ml  H200 + additives:  5 ml H1M, 20 ml H0, (62.5 ml 20% IPEGAL), 5 ml             0.5 M GTP, 1.7 ml BME, 25 ml protease inhibitors.

 

 

The Prep:

 

 

1.  Prepare 1:1 (w/v) 0-3hr embryo extract in HEPES Homogenization Buffer

 

            -blot embryos dry on sieve with paper towels before weighing.

            -homogenize with 5 passes of pestle in homogenizer.

 

2.  Spin extract 10 min, 3000 rpm in SS34.  Freeze extract in liquid nitrogen and store at -80° until ready to use.

 

3.  Thaw extract quickly in 37° water bath.  Transfer to 5-6 SW55 tubes on ice.  Spin 39,000 rpm in SW55 for 1.5 hrs, 4°, use deceleration 4 setting on ultra.

 

4.  Note clarified extract volume, divide into two tubes suitable for SS34 spin.

Add 30% PEG-8000 in H100 to clarified extract to final concentration of 2%.  Incubate of ice 30 min.

 

5.  Spin 20 min, 17,000 rpm, SS34.  Discard sup.

 

6.  On ice, resuspend in total (for both tubes) of 12-15 mls H200 + 0.05% IPEGAL (or NP40), 0.1 mM GTP, 1mM b-mercaptoethanol, 1:1000 protease inhibitors.

 

            -use pestle from a homogenizer to help resuspend.  Pipeting up and             down with a plastic pipet also helps.  Takes about 10 min on ice to             completely resuspend.

 

7.  Transfer to SW55 tubes.  Clarify in SW55, 35,000 rpm, 20 min.

 

8.  Transfer sup to 15 ml conical tube.  Add 6 ml C17, pH2 elution (1.3 mg/ml) antibody per ml of clarified sup.  Tumble slowly in cold room for 1 hr.

 

9.  Add 50-65 ml packed Affi-Prep Protein A beads (Bio-Rad) that have been pre-washed in 3 x 1ml H200 + 0.05% IPEGAL (or NP40), 0.1 mM GTP, 1mM b-mercaptoethanol, 1:1000 protease inhibitors.  Tumble slowly in cold room for 1 hr.

 

10.  Spin beads down in clinical centrifuge in cold room, top speed, 4-5 min.  Aspirate off sup, carefully avoiding beads.  Resuspend beads in 1 ml H200 + 0.05% IPEGAL (or NP40), 0.1 mM GTP, 1mM b-mercaptoethanol, 1:1000 protease inhibitors, transfer to a microfuge tube.  Spin 10 sec in Nanofuge to pellet beads.  Wash beads twice more in 1 ml same buffer.

 

11.  Wash beads 3 x 1ml in same H200 buffer without detergent.  Wash 3 x 1ml in H100 buffer.

 

12.  Resuspend beads in 50 ml 1 mg/ml C17 peptide in H100 + 0.1 mM GTP, 1mM b-mercaptoethanol, 1:1000 protease inhibitors.  Let sit on ice over night in cold room (no tumbling).  (I usually let it sit from about 3:30 pm to 9 am).

 

DAY 2

 

1.  Gently resuspend beads.  Pellet 10 sec in Nanofuge.  Transfer sup to a fresh tube.

 

2.  Wash beads gently with 2 x 50 ml elution buffer.  Save these washes also--they will have a lot of gTuRC in them.  Sometimes I combine the first wash with the overnight elution, depending on what I want to do with the prep.

 

3.  Wash the beads with 2 x 1 ml H100.  Boil beads in 100 ml 1X Sample Buffer for a gel.  Save 10 ml gTuRC for gel also.  Load 10 ml uneluted and eluted samples on gel for Coomassie staining.