Generally people prefer to process IP (Immuno ppt) cells immediately, but is there any fault if I freeze cells and lyse them later? Also can I use the same frozen lysate (kept after lysing cells in -80 deg freezer) for an IP experiment (processing for IP pull down) for further processes in the future?
It depends on what you'll be doing with the ippt'd proteins afterwards. If you don't need them to be active, freezing the samples prior to ippt shouldn't be a problem (it may even help break up your cells for making the lysate). If you do need the proteins to be active and not denatured, freezing may still be okay, but I'd suggest you do the experiment and verify that your protein(s) of interest aren't being significantly degraded by the freeze-thaw cycle.
Freezing is ok for many IPs but if you plan kinase assays after IP, its better to proceed with unfrozen lysates to get maximal activity (frozen lysates may still work, but with less efficiency). Certain proteins are very sensitive to freeze thaw cycles as they loose 3D folding (which might lead to loss of protein-protein interaction).
I don't see any harm in such freezing, though it looks rather pointless - cells are prone to disruption during freeze-thawing, so why not to lyse them first and then freeze the lysates? This extends the procedure for as little as 10 min (or 20 min if you clarify the lysates which is preferable). Otherwise you better minimize the cell medium volume to prevent dilution effects if cells get disrupted and their contents get free into solution. Or use some DMSO as suggested above.
Thanks all of you for your kind suggestions.. it will help me a lot..@Alexander , @Goodwin, @Michael , @ Bryan .
Hi Debasish,
Did you try to freeze the lysates and used for IP later? How did it work for you?
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