Cytosolic and nuclear fractionation
Reagents:
- Protease inhibitor cocktail from Roche, #11697498001.
- Dounce homogenizer with pestle A and B.
- 100 um mesh
This method is used to make nuclear and cytosolic fractions from mouse tissues including liver and heart. To monitor protein phosphorylation, phosphatase inhibitors are included in the extraction buffer.
1. The day of fractionation, make extraction buffer. For 10 ml you will need:
Hepes, 4mM, pH 7.4, 400 uL from stock (0.1 M stock: 2.38 g/100 ml) Sucrose, 320 mM, 1.094 g DTT, 1 mM, 10 ul from 1 M stock Na orthovanadate, 1 mM, 2 mg NaF, 8 mg MgCl2, 10 mM, 100 ul from 1 M stock KCl, 5 mM, 50 ul from 1 M stock EDTA Free mini Tab (protease inhibitors), 1 tab Triton x-100, 0.1%, 10 ul from 10% stock. Add last as it tends to foam
2. For 50 mg of tissue, add 250-500 ul of the extraction buffer and homogenize using the Dounce homogenizers (order- Pestle A, 20 strokes, followed by Pestle B of 20 strokes) on ice.
3. Carefully filter the homogenate using 100 um mesh (you might lose some volume). Seek assistance if needed.
4. Transfer flowthrough into Tube 1 (label Nuclear Pellet). Vortex it gently and remove 10 ul for protein assay (Tube 2-label for protein assay, containing 90 ul of 1 X TBS).
5. Also aliquot 40 ul into Tube 3 - label Total fraction, containing 40 ul of 2 X SDS-PAGE loading buffer.
6. The remaining volume (labeled Nuclear Pellet) is subjected to centrifugation at 2K RPm for 3 min at 4C.
7. Separate the supernatant carefully without disturbing the pellet into tube 4 – labelled soluble portion-initial. This leaves behind Tube 1 with nuclear fraction.
8. Processing of Nuclear and Cytosolic fractionations is as follows.
Nuclear
1. To process the Nuclear fraction, take tube 1 containing pellet, add ~ 500 ul of the extraction buffer and spin at 2K RPM for 3 min at 4C. Discard the supernatant.
2. Repeat washing the nuclear pellet for the second time using extraction buffer, and spin down at 2K RPM for 3 min at 4C. Discard the supernatant.
3. To the final pellet, add 200 ul extraction buffer and gently break the pellet by tapping.
4. Then add 200 ul of 2 X SDS-PAGE loading buffer to the pellet fraction (final volume 400 ul).
5. Pass it through the glass wool and aliquot into several tubes as desired.
6. Nuclear fraction is twice concentrated than the Cytosolic fraction.
Cytosolic
1. Centrifuge tube 4 (soluble portion-initial) for 10 min of 2K RPM at 4C.
2. Separate the supernatant without disturbing the smear-like pellet into Tube 5 (label soluble portion-final or Cytosolic fraction).
3. Measure the approximate amount of supernatant (~400 ul) and add the same volume of 2 X SDS-PAGE loading buffer (~400 ul).
4. To the soluble portion-initial pellet or tube 4, add 20 ul of the extraction buffer and 20 ul of 2 x SDS-PAGE loading buffer.
5. Determine the protein concentration and make aliquots of Nuclear and Cytosolic fractions (for western blots) in required concentrations.
In the end, you will have:
Tube 1: Nuclear fraction – aliquots saved @ -80C. Tube 2: For Protein assay – saved at -20C. Tube 3: Total Fraction – saved at -80C. Tube 4: Soluble Portion-Initial – saved at -20C. Tube 5: Cytosolic fraction – aliquots saved at -80C.
Protocol courtesy by Battiprolu P.