Betsy O'Neill

MAKING FOOTPRINTING EXTRACTS FROM DROSOPHILA HEADS


This is a protocol which I adapted from the standard Tjian lab embryo nuclear prep. I generally make extracts from 200-500g of adult flies at a time. I harvest them into disposable 250 ml Corning centrifuge tubes, and store them at -70 until I'm ready to use them (up to a few weeks at least). I separate the heads by placing the tubes into liquid nitrogen for a few minutes and then shaking the tubes vigorously. This causes the heads, wings and legs to fall off. I then put this material through two sieves-a number 25, which retains the abdomen and thorax, and then a number 40 which retains the heads, but allows the legs and wing fragments to go through. This also requires vigorous shaking to get good separation. I had an apparatus constructed as diagrammed below which I place into a multivortexer to acheive separation more easily (I had to do this because my arms got tired before I had acheived good separation).
footprinting extracts; head extracts



I then place the still frozen heads into about 1ml of high sucrose homogenizing buffer per 3g of adult flies. I put this mixture through a Yamato continuous flow homogenizer set at 700 rpm. I collect the flow through and pass it through the homogenizer another 4-5 times. I periodically examine samples of the flow through under a dissecting scope to monitor the efficency of homogenization. This makes it easy to determine when all of the heads have been broken up. Next, I pass the homogenate through prewetted (ddH20) miracloth (Calbiochem #475855) placed in a large funnel, which removes large particulate matter, but allows nuclei to pass through. Nuclei are then isolated by spinning at 24K for 45 minutes at 4 C in an SW28 rotor. Since the SW28 tubes must be filled completely for the spin, I usually bring the volume of the material that comes through the miracloth up to something divisible by 40ml, since the tubes hold about 38ml. I experimented initially with spinning through a sucrose cushion on this step (hence the SW28 rotor etc.), but had difficulty isolating the nuclei--they seemed to be at the interface rather than in the pellet as described in the protocol I was trying to adapt, so I eventually settled on the 24K spin. There is generally a large layer of lipid at the top of the tube after this spin, which I usually scoop off before attempting to pour off the cytoplasmic fraction.
The pellet (nuclei) from this spin is resuspended in 0.1ml of Buffer A+B per gram of flies. The pellet itself is usually composed of a softer upper layer, the nucei, and a hard, dark lower layer. I usually resuspend only the top layer and discard the lower layer. Transfer this material into Ti60 tubes. The nuclei are lysed (in the Ti60 tubes, so that there is no need to transfer the viscous solution containing lysed nuclei) by adding 0.1 volume of 4M ammonium sulfate pH8.0. I usually add this a little at a time, and mix well, but gently, in order to get even lysis. I then place the tubes on a rotating wheel at 4 C for 20 minutes. Pellet the chromatin by spinning at 35K for 30 minutes in a 60Ti rotor at 4 C. The supernatant from this spin is a >10% AS cut. The crude extract that I generally work with is a 10-60% AS fraction which I prepare by adding 0.3g of finely ground ammonium sulfate per ml of extract. We have a coffee bean grinder that I use to grind the AS, but I'm not sure that's necessary. I place the extract in a small beaker in an ice bath on a stir plate in the cold room, add the AS gradually, and stir for 15 minutes. I then transfer this material to a Sorval tube, and spin at 15K for 30 minutes. I resuspend the pellet in 2ml of 0.1M HEMG per 100g flies, and dialyze against 0.1M HEMG until the conductivity is equal to that of 0.1M HEMG (takes 4-6 hours). This is my final extract which I aliqout and freeze in liquid nitrogen, and then store at -70 C. The protein concentration of the extract is usually around 15 mg/ml, using BSA as a standard, or about 30mg protein per 100g flies. I have experienced quite a bit of variability in yield, which I think is due mainly to variation in the efficiency of the early steps (i.e., harvesting heads). For example, if you don't shake the frozen flies long enough to get all the heads off, the yield will be lower.
I see good footprints on the rhodopsin promoters using in the range of 10-100 ug extract in a standard footprinting reaction.

Protocol Summary:
1) Harvest heads from adult flies; by using appropriate sieves.

2) Place heads into a beaker containing 1 ml homogenizing buffer per 3g adult flies.

3) Homogenize in continuous flow homogenizer at 700 rpm. Collect flow through and rehomogenize until all heads are broken open.

4) Pass homogenate through prewetted miracloth to remove particulate matter.

5) Bring the total volume of this filtrate up to an amount divisible by 40 with homogenizing buffer, mix well, and transfer to Ultra Clear SW28 tubes (about 38 ml per tube). Spin at 24K/4 C/45 minutes.

6) Remove upper layer of lipid and pour off supernatant (cyto). Resuspend pellet (nuclei) in 0.1ml Buffer A+B per gram of adult flies.

7) Transfer this material (noting volume) to 60Ti tubes. Lyse nuclei by adding 1/10 volume 4 M ammonium sulfate pH 8.0. Add the AS a little at a time, and mix gently by capping the tube and inverting it several times. This should cause the mixture to become quite viscous.

8) Place the tubes on a rotating wheel at 4 C for 20 minutes.

9) Pellet the chromatin by spinning at 35K/4 C/30 minutes.

10) Place the supernatant from this spin (note the volume) into a small beaker containing a stir bar, and place the beaker in an ice bath on a stir plate in the cold room. Gradually add 0.3g finely ground ammonium sulfate per ml of supernatant to the stirring extract. Stir for an additional 15 minutes.

11) Transfer this material to a Sorval tube and spin 15K/4 C/30 minutes.

12) Resuspend the pellet in 2ml 0.1M HEMG per 100g adult flies and dialyze against 0.1M HEMG until the conductivity of the extract equals that of 0.1M HEMG.

13) Aliquot a portion of the extract into small aliquots, and then freeze in liquid nitrogen.

SOLUTIONS
additions per 1 liter
Buffer A+B ; 15mM HEPES pH 7.6 15 ml of 1M (pH w/ KOH)
100mM KCl 7.46g
5mM MgCl2 5ml of 1M
just before use add:
1mM DTT 1:1000 dilution of 1M
0.5mM sodium metabisulfite 1:1000 dilution of 95mg/ml
0.1mM PMSF 1:200 dilution of 0.2M
(in EtOH store at 4 C)
homogenizing buffer;
additions per 1 liter
Homogenizing 10mM HEPES pH 7.6 10 ml of 1M
Buffer 25mM KCl 1.87g
0.15mM spermidine 174 mg
0.5mM spermine 38 mg
1mM EDTA pH 7.9 2 ml of 0.5M
2M Sucrose 685g
10% glycerol 100ml
just before use add:
1mM DTT 1:1000 dilution of 1M
0.5mM sodium metabisulfite 1:1000 dilution of 95mg/ml
0.1mM PMSF 1:200 dilution of 0.2M

additions per 2 liters
0.1M HEMG; 25mM HEPES pH7.6 50 ml of 1M
100mM KCl 14.93g
12.5mM MgCl2 25 ml of 1M
0.1mM EDTA pH 7.9 0.4 ml of 0.5M
10% glycerol 200 ml
just before use add: (to 2 liters)
1mM DTT 2 ml of 1M
0.5mM sodium metabisulfite 190 mg
0.1mM PMSF 1 ml of 0.2M

4M ammonium sulfate
 Add 52.8g ammonium sulfate per 100 ml. Adjust the pH with NaOH. This solution is almost saturated at room temperature. Filter any undissolved crystals and store at room temperature.