I am going to isolate nuclear protein from ~30 tissues. The lysis buffer contains 10mM NaCl and 0.1% Triton X-100 which is reported with the ability to lyse cell membrane only (but not nuclear membrane). I will homogenize tissues with the lysis buffer and then leave them on ice, until I finish the homogenization of all 30 tissues. It may take a few of hours.
My question: How many minutes or hours can I put the lysate on ice without beaking nuclear membrane and releasing nuclear proteins BEFORE the spin to pellet the nuclei and cell debris?
Thank you for any idea.
Dear XianFeng, you could validate the lysis procedure by keeping a TX100 treated tissue sample on ice for various times and assay for a nuclear protein, e.g. by western blot or for an enzyme activity.
Alternatively, flash freeze each sample after homogenization in liquid nitrogen until further workup.
Nuclei are remarkably stable and neither buffer should lyse nuclei however proteins will begin to diffuse out of the nuclear pores given time. This is well known for certain spliceosome proteins. regardless, we routinely pipette off the buffer from the nuclei pellet and store them or freeze in -80 as a (relatively) dry pellet. Later we will take the nulcei and just add the nuclear lysis buffer. You can even wash in PBS before storing. There shouldn't be any reason to leave the nuclei in lysis buffer. Spin and removing lysis buffer will take 5 min. You can also confirm that your nuclei are not lysed and relatively "pure" by looking through a microscope. BTW TritonX or NP40 will lyse nuclei if you raise the salt concentration too much. Like 200 mM NaCl will cause nuclear lysis.