method of making nuclear extract for protein interaction? - (May/15/2006 )
hi dear all.
question again. not solved in my last post , so make it clear now.
What is the lysis buffer you guys use for making nuclear extract and this nuclear extract is used for making protein complex? anyone did this before? Due to the protein complex, so the method should be quite gentle to not to break the complex or lose any component, that is what I thought, but no clue which one is the best.
I use this protocol to Co-IP complexes from NE all the time.
# Add protease inhibitors to all buffers (below)
# Harvest cells and wash once in 1X PBS - Establish approx volume of pellet.
# Centrifuge cells and resuspend in 10 pellet vol of Hypotonic Buffer
# Allow cells to swell with 10 min incubation on ice
# Homogenize the cells 8 times with a 'loose' pestle - dounce homogenizer.
# Pellet the nuclei at 2000g for 10 min
# Resuspend the pellet in 0.5 pellet volumes (for new pellet size) of Low Salt Buffer. -----This is whole nuclear extract
# Homogenize the cells 6 times with a 'loose' pestle - dounce homogenizer.
# Slowly add 0.5 pellet volumes of High Salt Buffer (0.42M KCl final)
# Rotate the nuclei for 30min at 4°C
# Remove the insoluble material by centrifugation at 14000g for 15min at 4°C
# Save supernatant at 4°C, just in case. is in the insoluble pellet. [Here the insoluble material is the matrix portion of your NE if you wish to either use or discard this portion for some reason.]
10mM Tris pH7.4; 10mM KCl; 1.5mM MgCl2; 1mM DTT; protease inhibitors
Low Salt Buffer
20mM Tris pH7.4; 20mM KCl; 1.5mM MgCl2; 1mM DTT; 25% glycerol;0.2mM EDTA; protease inhibitors
High Salt Buffer
20mM Tris pH7.4; 1.2M KCl; 1.5mM MgCl2; 1mM DTT; 25% glycerol; 0.2mM EDTA;protease inhibitors