Things to do before you start. It is a good idea to take care of items 1 - 6 the day before.  All labware should be cleaned and rinsed throughly with nanopure water.

1. Turn on, chill and reserve Sorvall centrifuge and Beckman Ultracentrifuge.
   
   
2. Chill Sorvall SLA-1500, HB4 and SS34 rotors and Beckman Ti70 rotor.
   
   
3. Clean four 250 ml bottles for GSA, four 40 ml tubes (the ones without caps) for HB4, and four 1 x 3.5 polycarbonate Ti70 tubes with aluminum caps (model 355618).
   
   
4. Chill one 55 ml Wheaton homogenization vessel that functions with motorized teflon pestle. (Use the pestle that has blue tap on the metal shaft {replace the tape if it comes off}. This one seems to be slightly narrower in diameter and homogenizes the embryos more easily than the other teflon pestles.)
   
   
5. For every 50 grams of embryos, set-up a double layer of green miracloth in a funnel supported at the opening of a 500 ml erlenmeyer flask. Rinse with copious amounts of water. You will notice a small amount of frothing by the solution that collects in the erlenmeyer suggesting that there might be some form of wetting agent in the miracloth - this does not seem to cause any problems. Allow the water to drain as much as possible, empty the flask and store the entire set-up in the cold. I recommend covering the entire set-up with plastic wrap so it stays clean.
   
   
6. Have two embryo collection apparatuses available with the appropriate screens.
   
   
7. Prepare the following solutions just before starting the preparation. Buffer A, high sucrose buffer and nuclear suspension buffer can be prepared ahead of time and stored cold - it is the additional items that should be added fresh. The composition of these are provided at the end or the protocol. Note that PMSF dissolved in isopropanol as a 100 mM stock and DTT is dissolved in water as a 1 M stock - both are stored in the freezer.
• 500 ml of 50% chlorox/1% triton-X100/1% NaCl in a 1 liter plastic beaker, prepared at room temperature.
• 300 ml of buffer A supplemented with 42 ul of beta-mercaptoethanol, 1.5 ml of 100 mM PMSF, 48 mg Benzamidine HCl, 31.2 mg Sodium Bisulfite (both Benzamidine HCl and Sodium Bisulfite should be added as solid since solutions are not stable) - chill on ice.
• 60 ml of high sucrose buffer supplemented with 8.25 ul of beta-mercaptoethanol, 9.6 mg Benzamidine HCl, 6.24 mg Sodium Bisulfite, and 0.3 ml of 100 mM PMSF - chill on ice.
• 75 ml of nuclear suspension buffer supplemented with 75 ul of 1M DTT, 0.375 ml of 100 mM PMSF, 12 mg Benzamidine HCl, 7.8 mg Sodium Bisulfite, chill on ice.
  
  
8. Have available 10 ml of 4M Ammonium sulfate, pH 7.9.
   
   
9. One 500 ml beaker chilled on ice for embryo suspension.
   
   
10. 1 liter of 40 mM HGKE for dissolving the ammonium sulfate precipitated nuclear extract and for dialyzing the extract. 1 liter of solution is also sufficient to dialyze extract from 200 grams of embryos so this does not have to be increased when more than 100 grams of embryos are processed.
• For 1 liter of 40 mM HGKE, mix together:
• 20 ml 2X "1M HGKE"
• 480 ml 2X "0M HGKE"
• 499 ml Water
• This solution is put in a 1 liter beaker with a stir bar and set up on a stirrer in the cold room. Cover it with plastic wrap. Shortly before this solution is needed, it must be supplemented with PMSF, DTT, Benzamidine HCl, and Sodium Bisulfite. Don't add these agents before starting the preparation because they are unstable.
  
  
11. Dialysis tubing that will hold 5 to 10 ml of extract. Soak Spectrapor tubing in water for several hours before use.
    
    
12. Dewer flask of liquid nitrogen (basement of chemistry building)

1. Collect trays of 0-12 hour old embryos. Embryos that have been laid on grape plates for 12 hour periods are generally used. The plates are transferred every 12 hours to the cold room where the embryos can be stored for up to three days. While in the cold room, the trays of embryos are kept in a large plastic container covered with a lid so that they do not dehydrate.
   
   
2. Collection of the embryos is done by setting up a collection apparatus with a course Nitex screen to remove adults and a fine Nitex screen to trap embryos. The collection apparatus consists of three gray interlocking pieces of plastic pipe that support the screens. Check with someone who is familiar with the screens so you can be certain you are using the correct ones. One collection apparatus will usually handle the eggs from 18 to 20 grape plates. I prefer to set up two collection apparatuses and alternate between the two as I add suspensions of embryos so there is time for the liquid to drain.
   
   
3. Set up the collection apparatus in the large sink in the fly room where there is plenty of room to accommodate the grape plates. The embryos on the trays are wetted with water and then brushed into a plastic tub. Squirt water on the trays to rinse remaining eggs into the plastic tub. Try to avoid the strip of yeast paste. Empty the container of embryos into the collection apparatus after every four trays. You want to avoid having the eggs become anoxic. Throughout the collection period, intermittently splash tap water on the embryos in the collection apparatus. Be certain that the tap water is room temperature or less. Occasionally the tap water is warm in the summer. If this is the case, you will need to chill large flasks of it.
   
   
4. Set the grape plates aside in a large covered container. If they have not dried and cracked, wash them off for reuse when you have time later in the preparation. It is a lot easier to reuse the grape plates than to pour fresh ones. (Clean the trays at some point when you have a break in the procedure. To clean, scrape most of the residual yeast off the surface of the agar with a spatula. Under running water, rub the surfaces free of eggs, yeast and fly debris using gloved hands or a sponge. Store the washed trays in a covered container in the cold room. If the trays are dried and cracked, they should be discarded in a plastic trash bag and the top of the bag should be taped shut.)
   
   
5. After all the eggs have been transferred from the trays, rinse the trapped eggs through the course screen by squirting them with water. Remove the course screen and rinse the eggs with cold tap water. Carefully remove the upper pipe. Fold the screen in half so that it surrounds the eggs and gently squeeze the water out of the patty of embryos. Squeeze the folded screen and patty of embryos between several paper towels until the most of the water is out. Weigh the screen and patty. The screen weighs about 2 grams; the rest is embryos. Place the patty of eggs on ice.

1. Set up two collection apparatus in the sink next to the cold cabinet. These are the same as the apparatus used to collect embryos from the grape plates except that the course screen is not needed. Water from one of the bench faucets must also be available through a long tube; this is needed to rinse the embryos.
   
   
2. Immerse the patty of embryos in 500 ml of fresh 50% Chlorox/1% triton/1% NaCl for 3'. Gently stir the embryos with a glass rod or a magnetic stirrer.
   
   
3. Slowly pour equal amounts of the embryo/bleach suspension into each of the collection apparatuses. Try to keep the chamber above the screen from becoming more than 2/3 full. Once the entire suspension has been transferred to the chamber and the chamber is about 1/3 full, the embryos should be washed with water from the bench faucet. Turn the water on low and pinch the end of the tubing so a forceful spray of water can be applied to the embryos. Rinse the embryos and then gradually increase the flow of the water. Wash until all scent of chlorox is gone and the embryo suspension no longer foams. Rinse each pad of embryos with about 1 liter of nanopure water. Allow the water to drain. Remove the screen and pad of embryos. Then fold the screen over the pad and squeeze most of the water from the embryos. Finally, blot the pad of embryos dry with paper towels.
   
   
4. Put the embryos in a 500 ml beaker. Add 200 ml of cold buffer A (plus additives) and disperse the cake with a clean glass rod.
   
   
5. Prewet the miracloth in each filter with about 10 ml of buffer A, and discard the buffer that drains into the flask.
   
   
6. Homogenize 40 ml portions with a motorized teflon pestle. Keep the homogenizer cold during the strokes by surrounding it with ice in a plastic container. Set the motor at ~4.5 to 5 and proceed to break the embryos with 8 strokes. Pour each batch into the funnel containing the prewetted, doubled, miracloth filter assembled in the cold cabinet.
   
   
7. Stir the homogenate in the funnel with a glass rod to facilitate filtration. When enough of the lysate has passed through the miracloth, close up the top of the miracloth and gently squeeze the remaining solution through the cloth. Push from the top down so that you force the lysate through the base of the miracloth cone. Be careful, you don't want the retentate oozing out into the lysate (don't worry if a little bit does by accident.
   
   
8. Transfer equal amounts of lysate to two clean 250 ml bottles and spin for 10' at 1500 rpm in SLA-1500 rotor.
   
   
9. Pour supernatant, which contains the nuclei, into clean 250 ml bottles and discard the remaining pellet. Don't worry if some of the pellet is carried over to the nuclear suspension.
   
   
10. Collect nuclei by spinning for 20' at 6000 rpm in a SLA-1500 rotor. Immediately after the rotor stops spinning, remove the rotor lid and turn the bottles 180^o so that the nuclear pellets are towards the bottom side of the bottle. Remove one bottle at a time and keep it in an angled orientation so that the pellet is down. Carefully aspirate off the supernatant. First draw off the white layer at the surface. Then suck off supernatant by guiding the tip of the pipette down the side of the bottle that is opposite the side of the nuclear pellet. Keep the tip near the surface of the supernatant. When you are near the bottom, slowly tip the bottle from its angled orientation to a verticle orientation. Look through the opening of the bottle. While the aspirator is drawing off supernatant, the nuclear pellet will become visible. Because it is a loose pellet, a wave of nuclei will head towards the pipette tip. Stop aspirating when the leading edge of this wave gets near the pipette tip. Its a good idea to have someone with experience guide you through this step when you are doing it for the first time.
    
    
11. Thoroughly disperse and combine the two nuclear pellets in a final volume of 50 ml with buffer A (plus additives). Disperse the nuclei by triterating with a 10 ml pipette. Leave as much of the hard yellow yolk pellet behind but don't worry if some comes along with the nuclei.
    
    
12. Overlay 12.5 ml portions of nuclear suspension on 15 ml of high sucrose buffer (plus additives) in four 40 ml tubes for the HB4 rotor. With a glass rod, slightly stir the interface between the two layers so that the nuclei do not clog at this point during centrifugation. (For 200g of embryos, you will overlay 25 ml on top of each 15 ml cushion of high sucrose.)
    
    
13. Check that samples are balanced (this is critical for the HB4 rotor since its clearance in the centrifuge is minimal; adjust with high sucrose buffer) and then collect nuclei by centrifuging at 12,500 rpm for 30' in the HB4 rotor.
    
    
14. Aspirate off the white material that collects at the top and then carefully aspirate the remaining supernatant. Never pour off the supernatant. If there is not a distinguishable pellet of nuclei, use a pipette to remove the supernatant that remains near the bottom of the tube. Err on the side of leaving behind a little supernatant so you don't discard nuclei.
    
    
15. Thoroughly disperse and combine the two nuclear pellets in a total volume of 60 ml with nuclear suspension buffer (plus additives). Leave most of the hard yellow yolk pellet behind.
    
    
16. Distribute 15 ml portions into four, 1 x 3.5 in. polycarbonate bottles (model 355618) and add 1.5 ml of 4M Ammonium sulfate (pH 7.9) to each tube. Cap (black plugs should include O ring), place sideways in ice, and shake gently for 30'.
17. Centrifuge at 35,000 rpm in a Beckman Ti70 rotor for 1 hr. at 4^oC.
    
    
18. Transfer the supernatant to a 100ml graduated cylinder and record the volume. The supernatant can be poured from the centrifuge tubes into the cylinder, but be careful because the gelatinous pellet sometimes comes loose. Be prepared to trap the gelatinous pellet with a clean blue pipet tip.
    
    
19. Transfer the nuclear extract to a beaker (150 to 200 ml size) that has been surrounded by ice and positioned on a stirrer with a stir bar. The stir bar should span most of the diameter of the beaker so that the solid ammonium sulfate that is added next does not pile up at the perimeter of the beaker.
    
    
20. Slowly add (over a period of 3') 0.3 g of solid ammonium sulfate per ml of extract. Before weighing the ammonium sulfate, grind it to a fine powder with a porcelin mortar and pestle. Any excess ammonium sulfate can be returned to the stock bottle. The extract should become cloudy white near the end of adding the ammonium sulfate. After adding the ammonium sulfate, continue stirring for 30'. Be sure that there is enough ice to maintain a cold solution.
    
    
21. Pour equal portions of the ammonium sulfate precipitated mixture into 40 ml centrifuge tubes, check that the tubes are balanced, and finally collect protein precipitate by centrifuging at 12,500 rpm for 20' in HB4 rotor.
    
    
22. Discard supernatant by pouring it out of the tube. Use a pipette to remove residual liquid but don't dry out the pellet (this is protein, not DNA).
    
    
23. Dissolve the ammonium sulfate precipitate in 5 ml of 40 mM HGKEDPX by dispersing the pellet with a blue pipette tip and gently triterating material. Set the pipettor at 500 ul so lysate doesn't get sucked up against the pipettor. It will take 15' to 30' to completely dissolve the protein pellet. The 40 mM HGKEDPX is prepared by adding the following to the 1 liter of 40 mM HGKE which should already be set up in the cold room:
• 1 ml 100 mM PMSF
• 154.3 mg DTT
• 160 mg Benzamidine HCl
• 104 mg Sodium Bisulfite
  
  
24. Dialyze the extract in 1 liter of 40 mM HGKEDPX until the conductivity of the extract is equal to between 100 and 130 mM HGKE. This takes about 3 hours. If the extract is to be chromatographed on DEAE, it will need to be adjusted to 100 mM before chromatography. Precipitate that forms at this later time will need to be removed by centrifugation.
    
    
25. Centrifuge sample in HB4 rotor at 12,000 rpm for 20' to remove insoluble material. It is easy to forget this step because it falls near the end of a fairly long day of work.
    
    
26. Determine conductivity and protein concentration. If not done at this time, it should be done sometime later before any chromatography or experiments are done with the extract.
    
    
27. Aliquot supernatant into desired portions, flash freeze in liquid nitrogen, store at -75^oC.

1 liter Buffer A (1M sucrose, 4 mM MgCl₂, 0.1 mM EGTA, 10 mM HEPES pH 7.6)
Put approximately 500 ml of water in a 1 liter beaker and set stirring.
Add:

500 ml high sucrose buffer (1.75 M sucrose, 2 mM MgCl₂, 0.1 mM EGTA, 10 mM HEPES pH 7.6)
Put approximately 200 ml of water in a 1 liter beaker and set stirring.
Add:

500 ml Nuclear suspension buffer (0.3 M sucrose, 2 mM MgCl₂, 0.1 mM EGTA, 0.1 M NaCl, 10 mM HEPES pH 7.6)
Put approximately 200 ml of water in a 1 liter beaker and set stirring.
Add:

100 mM PMSF stocks are made by dissolving 174 mg of PMSF in isopropanol at a final volume of 10 ml. This is conveniently done in a 15 ml orange capped tube which has 1ml gradations on the side. Store the stock in the freezer. Some of the PMSF will precipitate during storage. Before use, completely dissolve the stock by heating it briefly to 37^oC and leaving it at room temperature during the extract preparation. Once in contact with water, the effective half life of the PMSF is only a few hours.

1M DTT stocks are made up in water and stored at -20^oC.

4M Ammonium Sulfate pH 7.9. Place 52.8 g of Ammonium Sulfate in a total volume of approximately 90 ml. Slowly add 10 M NaOH to adjust the pH to 7.9. The ammonium sulfate will not completely dissolve until the pH is properly adjusted and few crystals may remain until the solution is adjusted to a final volume of 100 ml. Be very patient when making up this solution. Store the solution at room temperature.

HGKEDPX buffer contains 25 mM HEPES pH 7.6 (adjusted with NaOH), 10% glycerol, 0.1 mM EDTA, 1 mM DTT, 0.1 mM PMSF, 1 mM Bisulfite, 1 mM Benzamidine HCl and potassium chloride as indicated (ie. 40mM HGKEDPX contains 40 mM KCl). The DTT, PMSF, Bisulfite and Benzamidine HCl (which correspond to "DPX" in the buffer name) are added just before use. Prepare 1 liter of 2X "OM HGKE" (50 mM HEPES pH 7.6, 20% glycerol, 0.2 mM EDTA) and 1 liter of 2X "1M HGKE" (2 M KCl, 50 mM HEPES pH 7.6, 20% glycerol, 0.2 mM EDTA). Filter each stock through nitrocellulose. These stocks are worth making because they compose the column buffers used in subsequent fractionation of the extract

1 liter 2X "OM HGKE" (50 mM HEPES pH 7.6, 20% glycerol, 0.2 mM EDTA)

• 200 ml glycerol
• 1 ml 0.2M EDTA pH 7.5-8.0
• 11.91 g HEPES
• 10 M NaOH to give a final pH of 7.6 (~ 2.5 ml)
• H₂O

• 149.12 g KCl
• 200 ml glycerol
• 1 ml 0.2M EDTA pH 7.5-8.0
• 11.91 g HEPES
• 10 M NaOH to give final pH of 7.6 (~ 2.3 ml)
• H₂O