Preparationofcrudenuclearextractfromratprimaryhepatocytes.docVIP

Preparation of crude nuclear extract from Mice Liver 1/6/06 Weight liver 100mg, cut into small pieces using a clean razor blade. Collect in a pre-chilled, clean 2ml Dounce homogenizer, dounce homogenize (10 times is enough) directly in 1ml of buffer A with protease inhibitors phosphatase inhibitors. Transfer to a 1.5ml of microcentrifuge tube. Vortex the tube vigorously on the highest setting for 15 seconds. Incubate the tube on ice for 10 minutes. Add 50ul of 10% NP to the each tube. Vortex the tube for 5 seconds on the highest setting. Incubate tube on ice for 1 minute. Vortex the tube for 5 seconds on the highest setting. Centrifuge the tube for 5 minutes at the maximum speed at 4?C. Immediately transfer the supernatant (cytoplasmic extract) fraction to a clean pre-chilled tube. Place this tube on ice until use or storage. Put the pellet produced in Step6 in liquid nitrogen 10 minutes. The samples can be stored at this step in -80?C. Resuspend the insoluble (pellet) fraction produced in Step6, which contains nuclei, in 500ul of ice-cold buffer B with protease inhibitors and phosphatase inhibitors. Vortex on the highest setting for 15 seconds. Return the sample to ice and continue vortex for 15 seconds every 10 minutes, for a total of 4-5 times. Centrifuge the tube at maximum speed (~16,000 x g) for 10 minutes at 4?C. Immediately transfer the supernatant (nuclear extract) fraction to a clean pre-chilled tube. Place on ice. Using BSA method to quantify the protein concentration using NER as blank. Prepare same amount protein by adding sample buffer for WB. Aliquot and store all extracts at -80°C until use. Buffer A: 200 ml Stock solution Volumn 10mM HEPES, PH 7.6 1M Hepes, Ph 7.6 2ml 10mM KCL 1 M KCl 2ml