Experiment 11 Isolation and purification of nuclei

1.Aim and request Master the principle and method of isolation and purification of nuclei from rat liver cells.

2.Principle The commonly used method for isolation and purification of nuclei involves the mechanical disruption and fractionation of cells into their different subcellular components. The sedimentation coefficient is different for all types of subcellular structures. The factors that determine the sedimentation coefficient for different components are the radius and the effective density of these microscopic objects. Therefore, we can isolate and purify such subcellular components from each other merely by centrifuging a mixture of them (a crude cell lysate) at different speeds.

3.Procedure ⑴ Put 4.5 ml fresh rat liver homogenate in a marked tube, and centrifugate at 4000 rpm for 20 min. Then transfer the supernatant to another tube for later use. ⑵ Add 1ml of 0.25mol/L sucrose-citric acid solution to the precipitation, and mix it softly with the glass- stick. This mixed solution is the nuclear suspension. ⑶ Put 5ml of 0.88mol/L sucrose-citric acid solution in another tube. Along the tube wall, spread with the pipette all the nuclear suspension slowly onto the liquid surface.

⑷ Centrifugate at 2000 rpm for 10 min, discard the supernatant. The precipitation is crude nuclei. ⑸ Dissolve the crude nuclei with 5ml nucleus wash. Centrifugate at 2000 rpm for 10min. Discard the supernatant. The precipitation is pure nuclei. ⑹ Dissolve the pure nuclei with 5ml NaOH solution for later use.

Step ml fresh rat liver homogenate centrifugate at 4000 rpm for 20 min supernatant precipitation Step 2 Add 1ml of 0.25mol/L sucrose-citric acid solution nuclear suspension For later use mix it softly with the glass- stick cytoplasmic component

Step3 5ml of 0.88mol/L sucrose-citric acid solution Add nuclear suspension (from step 2) Step4 Centrifugate at 2000 rpm for 10 min discard the supernatant crude nuclei

Step 5 Dissolve the crude nuclei with 5ml nucleus wash Centrifugate at 2000 rpm for 10min Discard the supernatant pure nuclei Step 6 Dissolve the pure nuclei with 5ml NaOH solution for later use.

4.Results Keep what is obtained from step 1 (cytoplasmic component) and step 6 (nuclear component) in store at room temperature, which will be used in the next experiment.