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Preparation of a Nuclear Protein Extract from Drosophila Embryos
Contributor: The Laboratory of Donald Rio at the University of California, Berkeley
This protocol describes the preparation of a nuclear protein extract from Drosophila embryos for analysis by Polyacrylamide Gel Electrophoresis and Western Analysis.
1. Rinse the embryos off the dish using Embryo Collection Solution to flood the dish and pour the Embryo Collection Solution and embryos into a 50 ml centrifuge tube.

2. Dechorionate the embryos using 50% Bleach.

3. Rinse with Embryo Collection Solution and then do a final rinse of the embryos with Embryo Rinse Solution.

4. Transfer the embryos to a microcentrifuge tube and let them settle. This protocol is designed for a volume of approximately 100 μl of embryos. Remove the Embryo Rinse Solution and rinse again with Embryo Rinse Solution.

5. Allow the embryos to settle again and remove the Embryo Rinse Solution.

6. Freeze the settled embryos in Liquid Nitrogen and store at -80°C.

7. Transfer 100 μl of embryos to the smallest Wheaton glass homogenizer possible and keep on ice in a cold room. Suspend the embryos in a small amount of Buffer I Plus Inhibitors to ease the transfer to the homogenizer, if so desired.

8. Add 400 μl of Buffer I Plus Inhibitors and keep on ice.

9. Homogenize using 7 to 10 strokes of pestle B (tight-fitting) on ice in a coldroom until the suspension becomes very milky.

10. Pour the homogenate into a microcentrifuge tube and centrifuge at 7,700 X g (approximately 9,700 rpm in a microcentrifuge) for 15 min at 4°C to pellet the yolk and nuclei.

11. Remove the supernatant-cytoplasmic extract and save it at -80°C.

12. Carefully remove the top white layer of the pellet, which contains the nuclei, with a P200 tip on a pipettor and return it to the rinsed out homogenizer. Avoid disrupting the bottom yellow yolk layer of the pellet.

13. Add 0.5 ml of Buffer I Plus Inhibitors to the nuclei and homogenize with 4 strokes of pestle B.

14. With a plastic transfer pipette, layer the homogenate on top of 0.5 ml of Buffer II Plus Inhibitors in a new microcentrifuge tube. The Buffer II Plus Inhibitors acts as a sucrose cushion.

15. Centrifuge at 1,310 X g (approximately 4,000 rpm in a microcentrifuge) for 30 min at 4°C.

16. Aspirate the supernatant. There should be a small pellet at the bottom and along the side of the tube.

17. Resuspend the pellet in 20 to 100 μl of 2X Sample Buffer (depending on the size of the pellet) by sonicating three times for 30 sec in a cup sonicator.

18. Store the extract at -20°C or -80°C. To prevent degradation due to freeze thaw cycles, aliquot the extract before freezing.

19. Run 1 μl of each extract on a Polyacrylamide mini-gel and silver stain the gel to quantitate the proteins (see Protocols on Casting Polyacrylamide Mini-gels and on Silver Staining).

Buffer I Plus Inhibitors   1 mM DTT
0.1 mM EDTA
15 mM HEPES-KOH, pH 7.6
10 mM KCl
5 mM MgCl2
100 μg/ml Phenylmethylsulfonyl Fluoride (PMSF) (CAUTION! see Hint #1)
4 μg/ml Pepstatin (CAUTION! see Hint #1)
10 mM Sodium Metabisulfite
2 μg/ml Aprotinin (CAUTION! see Hint #1)
0.5 mM EGTA
50 μg/ml Leupeptin (CAUTION! see Hint #1)
Add the following just before use:
0.35 M Sucrose
Buffer I   0.35 M Sucrose
15 mM HEPES-KOH, pH 7.6
0.5 mM EGTA
5 mM MgCl2
10 mM KCl
0.1 mM EDTA
Embryo Rinse Solution   0.7% (w/v) NaCl
50% Bleach   50% (v/v) Bleach (CAUTION! see Hint #1)
Embryo Collection Solution   0.7% (w/v) NaCl
0.04% (v/v) Triton X-100
Sample Buffer (2X)   0.25% (w/v) Bromophenol Blue
1.4 M 2-Mercaptoethanol
6% (w/v) SDS
100 mM Tris-Cl, pH 6.8
20% (v/v) Glycerol
Buffer II Plus Inhibitors   1 mM DTT
50 μg/ml Leupeptin
0.1 mM EDTA
15 mM HEPES-KOH, pH 7.6
4 μg/ml Pepstatin
10 mM KCl
5 mM MgCl2
0.8 M Sucrose
10 mM Sodium Metabisulfite
2 μg/ml Aprotinin
0.5 mM EGTA
Add the following just before use:
100 μg/ml PMSF
Buffer II   0.8 M Sucrose
15 mM HEPES-KOH, pH 7.6
0.5 mM EGTA
5 mM MgCl2
10 mM KCl
0.1 mM EDTA
BioReagents and Chemicals
Magnesium Chloride
Sodium Metabisulfite
Potassium Hydroxide
Potassium Chloride
Sodium Chloride
Nitrogen, Liquid
Bromophenol Blue
Triton X-100
Protocol Hints
1. CAUTION! This substance is a biohazard. Consult this agent's MSDS for proper handling instructions.