Gradient Centrifugation Based Purification of Nuclei: A Technique to Obtain Ultrapure Fraction of Intact Nuclei Using Density Gradient Centrifugation
Transcript
To isolate and purify intact nuclei, begin by taking a crude nuclei fraction in a tube. The fraction contains intact nuclei along with broken nuclei fragments and debris.
Mix this fraction with a suitable density gradient medium such that it forms a suspension of the lowest concentration.
Now, layer solutions of increasing densities of the same gradient medium beneath the pre-prepared crude nuclei fraction to generate a multi-step discontinuous density gradient. This step allows the crude nuclei fraction to form a band at the top of the gradient.
Next, centrifuge the tube at a low speed.
Based on the differences in shape and mass, intact nuclei migrate differently compared to debris and nuclear fragments. This results in intact nuclei forming a separate band.
After centrifugation, locate the distinct band consisting of intact nuclei at the interface of density gradient solutions.
Using a pipette, aspirate the top layers of the gradient to facilitate access to the pure nuclei band. Collect the nuclei-containing band into a fresh tube.
Visualize these nuclei under a light microscope. Intact nuclei appear perfectly round with an intact nuclear membrane surrounding them.
Finally, store the pure nuclear fraction for further analysis.
