Do you have a protocol for isolation of nuclei from mammalian cells??
I've used this kit before and it's pretty good.
There are different protocols to extract nuclei.
We noticed that different protocols will give you different results in terms of protein enrichment (you could obtain a lysate enriched in proteins linked to DNA but not in structural proteins). Try different protocols.
Anyway we use the following procedure:
NP-40 Lysis buffer:
0,5% Nonidet P-40, 0.5M Sucrose, 15mM Tris, pH 7.5, 60mM KCl, 0.25mM EDTA, 0.125mM EGTA, 0.5mM Spermine, 0,125mM Spermidine, 40mM NaF, 1mM Na3VO4, 1mM DTT, Protease inhibitor cocktail
Resuspend cells in NP-40 lysis buffer (ice cold). Wait 5 minutes and shake gently every minute.
Centrifuge for 5 minutes at 600g. Nuclei are in the pellet.
Wash pellets in NP-40 lysis buffer 3 times
thnks a lot! but we really dont have the finance to buy a kit! :(
You have given me only the composition of the buffer with NP 40. what is the protocol?? we usually spin at 5500g for 5 mins at 4C to collect the nuclei in a hypotonic solution of sucrose. The yeild of the number of nuclei isolated are very less. i need to isolate atleast 300 nuclei to continue the protocol designed. Please help me.
Use a hypotonic lysis buffer with milder detergent (up to 0.5%) and pH >7.5. Use protease inhibitors if required. Lyse gently on ice and collect pellet at 5000g for 15min and collect the pellet.
Sorry, but I had some problem with the text editor.
OK. Resuspend cells in NP-40 lysis buffer (1ml every T75cm2 flask) and leave on ice for 5min. Centrifuge cells at 600xg for 5 minutes. Then wash the pellet 3 times with NP-40 buffer and then lyse them in the lysis buffer you prefer.
Best results can be obtained previously disrupting cells with a Dounce homogenizer using Mitochondira Isolation Buffer - according to Sun et al (2006) "Localization of GRP78 to mitochondria under the unfolded protein resposnse". This step will pre-disrupt cells and will give you a better enrichment of nuclei fraction.
Thnks a lot for all the suggestions! i shall update you on the progress of my work. thnks a lot guys!